Multivalent pcv2 immunogenic compositions and methods of producing such compositions

ABSTRACT

An improved method for recovering the protein expressed by open reading frame 2 from porcine circovirus type 2 is provided. Also provided is recombinant PCV2 ORF2 protein, and immunogenic compositions comprising PCV2 ORF2 protein. Moreover, multivalent combination vaccines are provided which include an immunological agent effective for reducing the incidence of or lessening the severity of PCV2 infection, preferably PCV2 ORF2 protein, or an immunogenic composition comprising PCV2 ORF2 protein, and at least one immunogenic active component of another disease-causing organism in swine,

SEQUENCE LISTING

This application contains a sequence listing in paper format and incomputer readable format, the teachings and content of which are herebyincorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

One aspect of the present invention is concerned with the recovery of aprotein expressed by open reading frame 2 (ORF2) of porcine circovirustype 2 (PCV2). More particularly, the protein is a recombinant proteinexpressed by a transfected virus containing recombinant coding sequencesfor porcine circovirus type 2, open reading frame 2. Still moreparticularly, the transfected virus is permitted to infect cells ingrowth media and the protein expressed by open reading frame 2 isrecovered in the supernate, rather than from inside the cells. Even moreparticularly, the method involves the steps of amplifying the openreading frame 2 gene from porcine circovirus type 2, cloning thisamplified portion into a first vector, excising the open reading frame 2portion from this first vector and cloning it into a transfer vector,cotransfecting the transfer vector with a viral vector into cells ingrowth media, causing the cells to become infected by the viral vectorand thereby express open reading frame 2, and recovering the expressedrecombinant protein coded for by open reading frame 2 in the supernate.

In another aspect, the present invention is concerned with animmunogenic composition effective for inducing an immune responseagainst PCV2, and methods for producing those immunogenic compositions.More particularly, the present invention is concerned with animmunological composition effective for providing an immune responsethat protects an animal receiving the composition and reduces, orlessens the severity, of the clinical symptoms associated with PCV2infection. Still more particularly, the present invention is concernedwith a protein-based immunological composition that confers effectiveprotection against infection by PCV2. Even more particularly, thepresent invention is concerned with an immunological compositioncomprising ORF2 of PCV2, wherein administration of PCV2-ORF2 results inprotection against infection by PCV2. Most particularly, the presentinvention is concerned with an immunological composition effective forconferring effective immunity to a swine receiving the immunologicalcomposition, and wherein the composition comprises the protein expressedby ORF2 of PCV2.

In another aspect of the present invention, combination vaccines ormultivalent vaccines are provided. More particularly, the presentinvention provides immunogenic compositions effective at inducing animmune response against infection by PCV2 and at least one otherdisease-causing organism for swine.

2. Description of the Prior Art

Porcine circovirus type 2 (PCV2) is a small (17-22 nm in diameter),icosahedral, non-enveloped DNA virus, which contains a single-strandedcircular genome. PCV2 shares approximately 80% sequence identity withporcine circovirus type 1 (PCV1). However, in contrast with PCV1, whichis generally non-virulent, swine infected with PCV2 exhibit a syndromecommonly referred to as Post-weaning Multisystemic Wasting Syndrome(PMWS). PMWS is clinically characterized by wasting, paleness of theskin, unthriftiness, respiratory distress, diarrhea, icterus, andjaundice. In some affected swine, a combination of all symptoms will beapparent while other swine will only have one or two of these symptoms.During necropsy, microscopic and macroscopic lesions also appear onmultiple tissues and organs, with lymphoid organs being the most commonsite for lesions. A strong correlation has been observed between theamount of PCV2 nucleic acid or antigen and the severity of microscopiclymphoid lesions. Mortality rates for swine infected with PCV2 canapproach 80%. In addition to PMWS, PCV2 has been associated with severalother infections including pseudorabies, porcine reproductive andrespiratory syndrome (PRRS), Glasser's disease, streptococcalmeningitis, salmonellosis, postweaning colibacillosis, dietetichepatosis, and suppurative bronchopneumonia.

Open reading frame 2 (ORF2) protein of PCV2, having an approximatemolecular weight of 30 kDa when run on SDS-PAGE gel, has been utilizedin the past as an antigenic component in vaccines for PCV2. Typicalmethods of obtaining ORF2 for use in such vaccines generally consist ofamplifying the PCV2 DNA coding for ORF2, transfecting a viral vectorwith the ORF2 DNA, infecting cells with the viral vector containing theORF2 DNA, permitting the virus to express ORF2 protein within the cell,and extracting the ORF2 protein from the cell via cell lysis. Theseprocedures generally take up to about four days after infection of thecells by the viral vector. However, these procedures have a disadvantagein that the extraction procedures are both costly and time-consuming.Additionally, the amount of ORF2 recovered from the cells is not veryhigh; consequently, a large number of cells need to be infected by alarge number of viral vectors in order to obtain sufficient quantitiesof the recombinant expressed protein for use in vaccines and the like.

Current approaches to PCV2 immunization include DNA-based vaccines, suchas those described in U.S. Pat. No. 6,703,023. However, such vaccineshave been ineffective at conferring protective immunity against PCV2infection and the clinical signs associated therewith.

Porcine Reproductive and Respiratory Syndrome (PRRS) is caused by avirus which was first isolated and classified as an arterivirus asrecently as 1991. The disease syndrome had been first recognised in theUSA in the mid 1980's and was called “mystery swine disease”. It hasalso been called blue ear disease. The name porcine arterivirus has beenproposed recently. The virus of PRRS has a particular affinity for themacrophages particularly those found in the lung. Macrophages are partof the body defences. Those present in the lung are called alveolarmacrophages. They ingest and remove invading bacteria and viruses butnot in the case of the PRRS virus. Instead, the virus multiplies insidethem producing more virus and kills the macrophages. Once it has entereda herd it tends to remain present and active indefinitely. Up to 40% ofthe macrophages are destroyed, which removes a major part of the bodiesdefence mechanism and allows bacteria and other viruses to proliferateand do damage. A common example of this is the noticeable increase inseverity of enzootic pneumonia in grower/finisher units when they becomeinfected with PRRS virus. It may take up to a year for all breedingstock, particularly in large herds, to become infected for the firsttime and although the virus appears to spread rapidly in a herd, it maybe some 4-5 months before at least 90% of the sows have becomesero-positive. Some sows remain naive. Furthermore, it is not uncommonfor sow herds 1-2 years after infection to contain less than 20% ofserological positive animals. This does not, however, necessarily meanthey are not still immune nor does it mean that they have stoppedpassing on immunity to their offspring. Adult animals shed virus formuch shorter periods of time (14 days) compared to growing pigs whichcan excrete it for 1-2 months. The clinical picture can varytremendously from one herd to another. As a guide, for every three herdsthat are exposed to PRRS for the first time one will show norecognisable disease, the second would show mild disease, and the thirdmoderate to severe disease. The reasons for this are not clearlyunderstood. However the higher the health status of the herd, the lesssevere the disease effects. It may be that the virus is mutating as itmultiplies, throwing up some strains that are highly virulent and somethat are not. PRRS infects all types of herds, including high orordinary health status and both indoor and outdoor units, irrespectiveof size.

Mycoplasma hyopneumoniae (M. hyo) is a small bacterium (400-1200 nm)classified in the mycoplasmataceae family. M. hyo is associated withEnzootic Pneumonia, a swine respiratory disease commonly seen in growingand finishing pigs. M. hyo attacks the cilia of epithelial cells of thewindpipe and lungs, causing the cilia to stop beating (ciliostasis) andeventually causing areas of the lungs to collapse. Depending on theextent of the disease, daily live weight gain of infected swine can bereduced by up to 17%. Enzootic Pneumonia is widespread in swinepopulations and present in almost every swine herd. M. hyo is consideredto be a primary pathogen that facilitates entry of PRRSV and otherrespiratory pathogens into the lungs. Three separate strains, 232, J &7448, have had their genomes sequenced (Minion et al., J. Bacteriol.186: 7123-33, 2004; Vasconcelos et al., J. Bacteriol. 187: 5568-77,2005).

Porcine proliferative enteritis is a common diarrheal disease ofgrowing-finishing and young breeding pigs characterized by hyperplasiaand inflammation of the ileum and colon. It often is mild andself-limiting but sometimes causes persistent diarrhea, severe necroticenteritis, or hemorrhagic enteritis with high mortality. The etiology isthe recently classified intracellular bacterium Lawsoniaintracellularis. The organism has been cultivated only in cell cultures,and attempts to propagate it in cell-free medium have failed. Koch'spostulates have been fulfilled by inoculation of pure cultures of L.intracellularis into conventionally reared pigs; typical lesions of thedisease were produced, and L. intracellularis was reisolated from thelesions. The more common, nonhemorrhagic form of the disease oftenaffects 18- to 36-kg pigs and is characterized by sudden onset ofdiarrhea. The feces are watery to pasty, brownish, or faintly bloodstained. After ˜2 days, pigs may pass yellow fibrinonecrotic casts thathave formed in the ileum. Most affected pigs recover spontaneously, buta significant number develop chronic necrotic enteritis with progressiveemaciation. The hemorrhagic form is characterized by cutaneous pallor,weakness, and passage of hemorrhagic or black, tarry feces. Pregnantgilts may abort. Lesions may occur anywhere in the lower half of thesmall intestine, cecum, or colon but are most frequent and obvious inthe ileum. The wall of the intestine is thickened, and the mesentery maybe edematous. The mesenteric lymph nodes are enlarged. The intestinalmucosa appears thickened and rugose, may be covered with a brownish oryellow fibrinonecrotic membrane, and sometimes has petechialhemorrhages. Yellow necrotic casts may be found in the ileum or passingthrough the colon. Diffuse, complete mucosal necrosis in chronic casescauses the intestine to be rigid, resembling a garden hose.Proliferative mucosal lesions often are in the colon but are detectedonly by careful inspection at necropsy. In the profusely hemorrhagicform, there are red or black, tarry feces in the colon and clotted bloodin the ileum.

Bovine Viral Diarrhoea Virus (BVD) and Border's Disease are two viruses,which are in the same group of pestiviruses as the virus of swine fever(hog cholera) but which primarily infect cattle and sheep respectively.They can get into pig breeding herds and cause reproductive problems.The disease is not a common cause of infertility in the sow and would beconsidered low on the list of possibilities from a diagnostic point ofview.

Leptospirosis is a contagious disease of animals, including man, causedby various immunologically distinct leptospiral serovars, most of whichare regarded as subgroups of Leptospira interrogans. There are fiveserovars and groups which are important in swine: pomona, australis,tarassovi, canicola, icterohaemorrhagicae, and grippotyphosa. Infectionsmay be asymptomatic or cause various signs, including anorexia, pyrexia,listlessness, jaundice, abortions, stillbirths and other vaguereproductive problems, and death. After acute infection, leptospiresfrequently localize in the kidneys or reproductive organs consisting ofscattered small grey foci of a focal interstitial nephritis, and areshed in the urine, sometimes in large numbers for months or years.Because the organisms survive in surface waters for extended periods,the disease is often waterborne. In the USA, the disease is primarilydue to the serovars Leptospira hardjo, Leptospira pomona, and Leptospiragrippotyphosa. Diagnosis can be difficult because antibody titers can betransient, lasting less than a month. Further, Leptospira can also befound in healthy animals. L. australis serovar bratislava is mostcommonly associated with reproductive problems. Chronically infectedherds display abortions, still births and weak piglets.

Brucellosis is caused by bacteria of the genus Brucella and ischaracterized by abortion, retained placenta, infertility, orchitis inboars, and severe metritis in sows. In piglets, the disease ischaracterized by posterior paralysis and lameness. The disease in pigsis caused almost exclusively by Brucella suis biovars 1, 2, and 3. Anumber of other mammals can carry and transmit Brucella suis to pigs.Infection spreads rapidly and causes many abortions in unvaccinatedherds. Transmission occurs mainly by contact with another pig, althoughvenereal transmission is possible. Serological diagnosis can bedifficult due to a relatively common organism, Yersinia enterocolitica0:9 which shares a common antigen with Brucella and often causes falsepositive results. Post-mortem lesions usually include metritis andorchitis, and can include abscessation, sometimes with necorsis foci inthe liver.

Clostridium is a ubiquitous gram-positive bacteria, of the familyclostridiaceae, usually found in the soil, but also occurs naturally inthe gut of most animals C. difficile infections in swine arecharacterized by severe mesocolonic edema, diarrhea and edema in othertissues such as the hydrothorax. Clostridium enteritis in swine iscaused by C. perfringens, and is characterized by chronic enteritis,which is accompanied by diarrhea, weight loss and fever. Infection withC. perfringens types A, B and C causes severe enteritis, dysentery,toxemia, and high mortality in young calves. Types B and C both producethe highly necrotizing and lethal β toxin that is responsible for thesevere intestinal damage. This toxin is sensitive to proteolyticenzymes, and disease is associated with inhibition of proteolysis in theintestine. Sow colostrum, which contains a trypsin inhibitor, has beensuggested as a factor in the susceptibility of young piglets. Thedisease can cause sudden death in piglets less than one week old, and ismost common within 3 days of birth. In older piglets, Clostridiumenteritis causes a thickening of the small intestine making absorptionof food and nutrients difficult. Piglets usually die as a result of acombination of the infection and lack of nutrients. Death may occur in afew hours, but less severe cases survive for a few days, and recoveryover a period of several days is possible. Hemorrhagic enteritis withulceration of the mucosa is the major lesion in all species. Grossly,the affected portion of the intestine is deep blue-purple and appears atfirst glance to be an infarction associated with mesenteric torsion.Smears of intestinal contents can be examined for large numbers ofgram-positive, rod-shaped bacteria, and filtrates made for detection oftoxin and subsequent identification by neutralization with specificantiserum.

Clostridium novyi has been suspected but not yet confirmed as a cause ofsudden death in cattle and pigs fed high-level grain diets, and in whichpre-existing lesions of the liver were not detectable. The lethal andnecrotizing toxins (primarily a toxin) damage hepatic parenchyma,thereby permitting the bacteria to multiply and produce a lethal amountof toxin. Usually, death is sudden with no well-defined signs. Affectedanimals tend to lag behind the herd, assume sternal recumbency, and diewithin a few hours. Most cases occur in the summer and early fall whenliver fluke infection is at its height. The disease is most prevalent in1- to 4-yr-old sheep and is limited to animals infected with liverflukes. Differentiation from acute fascioliasis may be difficult, butperacute deaths of animals that show typical lesions on necropsy shouldarouse suspicion of infectious necrotic hepatitis. The mostcharacteristic lesions are the grayish yellow necrotic foci in the liverthat often follow the migratory tracks of the young flukes. Other commonfindings are an enlarged pericardial sac filled with straw-coloredfluid, and excess fluid in the peritoneal and thoracic cavities.Usually, there is extensive rupture of the capillaries in thesubcutaneous tissue, which causes the adjacent skin to turn black (hencethe common name, black disease).

Clostridium septicum is found in soil and intestinal contents of animals(including man) throughout the world. Infection ordinarily occursthrough contamination of wounds containing devitalized tissue, soil, orsome other tissue-debilitant. Wounds caused by accident, castration,docking, insanitary vaccination, and parturition may become infected.General signs, such as anorexia, intoxication, and high fever, as wellas local lesions, develop within a few hours to a few days afterpredisposing injury. The local lesions are soft swellings that pit onpressure and extend rapidly because of the formation of large quantitiesof exudate that infiltrates the subcutaneous and intramuscularconnective tissue of the affected areas. Accumulations of gas areuncommon Malignant edema associated with lacerations is characterized bymarked edema, severe toxemia, and death in 24-48 hr.

Tetanus toxemia is caused by a specific neurotoxin produced byClostridium tetani in necrotic tissue. Almost all mammals, includingswine, are susceptible to this disease. Although tetanus is worldwide indistribution, there are some areas, such as the northern Rocky Mountainsection of the USA, where the organism is rarely found in the soil andwhere tetanus is almost unknown. In general, the occurrence of C. tetaniin the soil and the incidence of tetanus in man is higher in the warmerparts of the various continents. Clostridium tetani, an anaerobe withterminal, spherical spores, is found in soil and intestinal tracts. Inmost cases, it is introduced into the tissues through wounds,particularly deep puncture wounds, that provide a suitable anaerobicenvironment.

Infection with Salmonella spp can produce diarrhea in animals of allages, especially those that are stressed, closely stocked, or exposed toa heavily contaminated feed or water supply. Salmonellosis is caused bymany species of salmonellae and characterized clinically by one or moreof three major syndromes—septicemia, acute enteritis, and chronicenteritis. The incidence has increased with the intensification oflivestock production. Although various types of Salmonella can causeinfections in pigs the classic salmonellas found in swine are S.choleraesuis and S. typhimurium. Their resulting clinical patterns ofmost salmonella are not distinct and different species of salmonellaetend to differ in their epidemiology. Plasmid profile anddrug-resistance patterns are sometimes useful markers for epidemiologicstudies. Septicemic salmonellosis is often associated with S.choleraesuis. Infected piglets demonstrate a reluctance to move,anorexia, a high fever of 40.5 C-41.6 C, and may have a shallow cough.Piglets may also be found dead with cyanotic extremities. S.choleraesuis is one of the rare diseases that can cause both pneumoniaand diarrhea and mortality of infected piglets is often high.Enterocolitis is generally associated with the more common S.typhimurium. Infections are characterized by yellow or watery diarrheathat may contain blood or mucus as the infection progresses. Mortalityis low and often associated with dehydration and potassium deficiencyfrom the diarrhea. Feces of infected animals can contaminate feed andwater, fresh and processed meats from abattoirs, plant and animalproducts used as fertilizers or feedstuffs, pasture and rangeland, andmany inert materials. Although S. choleraesuis is rarely found in feed.It can also be passed directly through contact with an infected animalSalmonella can survive for months in wet, warm areas such as in feederpig barns or in water dugouts. Rodents and wild birds also are sourcesof infection. The prevalence of infection varies among species andcountries and is much higher than the incidence of clinical disease,which is commonly precipitated by stressful situations such as suddendeprivation of feed, transportation, drought, crowding, parturition, andthe administration of some drugs.

Escherichia coli is a bacteria of the enterbacteriaceae family and isone of the main types of bacteria naturally occurring in the smallintestines of all mammals. Although usually harmless, some E. colistrains can produce a number of exo- and endotoxins that cause infectionand disease. Heat-labile (LT) and heat-stable (ST) exotoxins areactively produced by some strains and are responsible for causing scour.Shigela-like toxin type II variant (SLT-IIe), Stx2e and verotoxin edemadisease act on the wall of the small arteries resulting in oedema.Endotoxins, such as Lipid A, play a role in mastitis and urinary tractinfections. E. coli infection is characterized by a number of differentsymptoms depending on the particular strain involved, includingdiarrhea, sunken eyes, unthriftiness, visible weight loss, stuntedgrowth, depression, bowel edema, mastitis, cystitis, pyelonephritis anddeath. E. coli can be classified and coded by their cell wall (Oantigens) and fimbriae (F antigens). For example, scour is oftenassociated with E. coli Abbotstown: O147, F4, F5, whereas bowel edema isassociated with F18 fimbriae. Correctly identifying the code isessential to the selection of the correct vaccine. E. coli infectionscompromise a pig's immune system and deaths are often the result ofsecondary infections and disease.

Swine Pox is a disease which causes skin lesions, paules, pustules andscabs.

Eperythrozoonosis is a Rickettsial (haemotrophic) disease caused byEperythrozoon suis, an extracellular bacterial organism that adheres topig erythrocyte membranes, inducing its deformation and damage. Thedisease is characterized by anemia and icterus (yellow discoloration ofmucous membranes, sclera and inner ears). It can lead to poor conceptionrates, other vague reproduction problems, and even death.

Hog cholera also known as Classical Swine Fever (CSF) or African SwineFever (ASF) is a disease caused by a Flaviviridae virus, which is anenveloped RNA virus, or in the case of ASF, an enveloped DNA virus thatis related to the Pox viruses. Clinically, CSF and ASF areindistinguishable. The first symptoms are a decrease in activity anddrowsiness with some anorexia and the swine may appear chilled. Withindays, pigs present with a marked fever (41-42 degrees Celsius), which issometimes accompanied by a reddening of the skin. Next, pigs develop aconjunctivitis and constipation leading to yellowish diarrhea. In herds,the pigs will appear chilled and will often huddle together. A few pigsmay convulse before dying. Pigs start to die with a spreading purplediscoloration of the skin and death often occurs within 10-20 dayspost-infection. Surviving pigs will oftentimes be affected by a severeretardation of their growth and arched backs. In established herds,piglets infected from their mothers during pregnancy can result inabortion, mummification, malformations, still births and weak bornpiglets. Piglets born from CSF-infected mothers may remain healthy butcontinually spread the disease throughout their lives.

Pneumonic pasteurellosis and Streptococci are caused by Pasteurellamultocida and various species of streptococci, typically S. suis.Infection by the causal agent generally represents the final stage ofthe post-weaning respiratory syndrome. Clinical signs appear in threeforms, the acute form is most commonly associated with P. multocidaserotype B. animals present with dyspnoea, labored breathing, thumping,high fever (42.2 Celsius), prostration, and finally death. In some casesthe abdomen becomes purple with discoloration. A second form is asub-acute form it is characterized by pleuritis, coughing, anddifficulty in breathing. Pigs can loose significant amounts of weightand may have poor or no growth with serious consequences in pig flow.The chronic form presents with the occasional cough, thumping, andlittle or no fever. This form generally affects pigs from 10-16 weeks ofage.

Streptococcal meningitis causes inflammation of the meninges which arethe membranes covering the brain. In the sucking piglet it is usuallycaused by Streptococcus suis, Haemophilus parasuis, or sometimesbacteria such as E. coli and other streptococci. S. suis has manyserotypes. In most countries S. suis type 1 is the main one in suckingpiglets, but this may not be true in other countries. For example inDenmark it is type 7. S. suis also causes joint problems particularlytypes 1 and 14. S. suis is carried for long periods in the tonsils andmay be transmitted to the sucking piglet from the sow or from otherpiglets. The sow also provides a variable level of immunity in thecolostrum. Streptococcal meningitis in sucking piglets is sporadic inindividual piglets. Streptococcal meningitis may be worse in suckingpigs when the organism has been introduced into the herd for the firsttime, or where it is secondary to infection with PRRS.

Pseudorabies, also known as porcine rabies virus, Suid herpes virus inwhich the causal agent is an enveloped herpes DNA virus. In naïve herds,neonatal pigs present with a range of severe central nervous signs fromfitting to severe in coordination. Posterior paralysis may result inpiglets sitting in a manner that resembles dogs. Additionally, mortalityis high. In weaned pigs, the central nervous signs may be reduced butmay be accompanied by an increase in respiratory signs. Oftentimes,respiratory diseases are associated with secondary infections. Weanedpigs can waste and suffer ill thrift and are often stunted. In growingpigs, the central nervous signs continue to reduce while the respiratorysigns increase. The degree of respiratory disease depends on thepresence and severity of secondary infections. In adults, reproductivesigns predominate. Sows may abort and animals infected close to term arelikely to give birth to stillborn or weak piglets. In established herds,there may be few clinical signs.

Swine Influenza Virus causes swine flu and belongs to the influenza TypeA virus group. In naïve herds, clinical signs may present in explosiveoutbreaks with all or many animals becoming ill at the same time.Animals may present with inactivity, depression, huddling/pilling andanorexia. The animals are often mouth-breathing and breathing islabored. Coughing may ensue upon movement. Other clinical signs includea nasal discharge and puffy eyes with rectal temperatures between40.5-41.5° Celsius. The high temperatures in a breeding stock can resultin abortions, infertility, production of small weak litters, andincreased still births. In established herds, annual reinfectionappears.

Spirochaetal colitis is caused by the Brachyspira pilosicoli bacteria.This infection generally effects 10-20 week old growers/finishers. It ischaracterized by a non-fatal wasting diarrhea of growing pigs thatresults in an increased number of days needed to finish. The diarrheaalso results in reduction in feed efficiency and produces waterydiarrhea or loose stools. About half of the pigs may show transient topersist to watery to mucoid green to brownish diarrhea without blood.The clinical signs are more common 10-14 days after mixing and changingof the feed.

Swine dysentery is caused by the bacteria Brachyspira hyodysentheriae.There are twelve known sero-types at this time. Clinical signs inestablished herd include diarrhea, a rapid loss of condition in somepigs, a hairy appearance, dehydration, painful abdomen, and the death ofone or two pigs before other pigs show any signs. In a key outbreak innaïve herds, all age groups from suckling piglets to adult sows can beeffected.

Transmissible gastroenteritis is a disease of the intestines caused by acoronavirus. It is in the same family as porcine respiratorycoronavirus, epidemic diarrhea virus, and hemagglutinatingencephalomyelitis virus. Initial clinical signs are watery diarrhea,vomiting, and anorexia. Piglets less than 21 days of age generally die,weaners become unthrifty, while growers, finishers, and adults aregenerally mildly affected and will survive if provided with adequatewater.

Parvovirus is a disease characterized by reproductive problems in pigs.The causal agent is a small DNA non-enveloped virus. Fetuses are theonly affected group and the effect on the fetus depends upon the age atwhich it becomes infected. At 10-30 days of age, infection results indeath and reabsorbtion of the fetus. Between 30-70 days of age,infection results in death and mummification. And from 70 days to term,infections results in the birth of weak piglets and mummification. Thedisease is able to cross the placenta and then move to each fetus alongthe uterus. In the sow, the clinical signs are still births, mummifiedpiglets, embryonic deaths, infertility, and the production of asignificantly reduced number of live-born offspring. Abortion is not acharacteristic feature of parvovirus infection.

Actinobacillus pleuropneumonia, also known as APP and Haemophiluspleuropneumonia, is caused by the Actinobacillus pleuopneumoniabacteria. There are currently 15 serovirus described and the severity ofthe clinical signs differ between the different serovirus and thepresence of other factors. Serovirus 1, 5, 9, 10, and 11 are consideredto be more virulent. Additionally, serovirus 1, 9, and 11; 2, 6, and 8;and 4 and 7 may cross-react. Pigs of all ages are susceptible. Clinicalsigns are a sudden illness that results in animals lying down a lot andpresenting a high rectal temperature of 41.5° Celsius. Animals aregenerally anorexic and do not drink, their extremities become cyanoticand cold to the touch. Cyanosis can spread to the whole body and severebreathing difficulties, often with mouth breathing, develop beforedeath. Blood-stained froth can be seen at the mouth and nostrils anddeath generally occurs within 24-48 hours. Acute clinical signs includea high percentage of animals in a group being depressed and lying down,high rectal temperatures of 40.5-41° Celsius, anorexia, lack ofdrinking, severe respiratory distress, coughing, mouth breathing,cyanosis, vomiting, and abortion. Sub-acute clinical signs includeintermittent coughing in a group of pigs, a general loss of appetite,and a reduction in growth. Cyrovar type 3 presents with arthritis,endocarditis, and abscesses. In chronically effected herds, daily weightgain may not be affected, but an intermittent cough may be heard.

Glässers Disease is caused by the bacterium Haemophilus parasuis (Hps),of which there are at least fifteen different types. It is foundthroughout the world and organisms are present even in high healthherds. If such herds are set up using SPF or MEW techniques and are freefrom Hps, it can be devastating when they first become contaminated,producing an anthrax-like disease with high mortality in sows. In themajority of herds in which the bacterium is endemic, sows produce astrong maternal immunity which normally persists in their offspringuntil 8 to 12 weeks of age. As a result, the effects of the infection inweaners are usually nil or minimal Disease may however be seen insuckling pigs. Pigs usually become sub-clinically infected when stillprotected by maternal antibody and then stimulate their own immuneresponse. If however, the maternal immunity wears off before they becomeinfected they may develop severe disease. This is usually sometime afterweaning. It can also act as a secondary pathogen to other major diseasesparticularly enzootic pneumonia (EP) (Mycoplasma hyopneumoniae).Outbreaks of disease are sometimes experienced in sucking pigs,particularly in gilt herds. Hps attacks the smooth surfaces of thejoints, coverings of the intestine, lungs, heart and brain causingpneumonia, heart sac infection, peritonitis and pleurisy. It isrespiratory spread. Disease caused by Hps is rare in sows unless the drysow is naïve. Lameness or stiffness, slight swellings over the jointsand tendons, and rarely meningitis, are occasionally seen in gilts. Inpiglets, acute disease presents with rapidly depressed pigs withelevated temperature, inappetence, and a reluctance to rise. Onecharacteristic feature is a short cough of 2-3 episodes. Sudden death ingood sucking piglets is not uncommon Hps is also known to causeindividual cases of arthritis and lameness with fever and inappetence.Chronic disease is characterized by pale and poor growing pigs. Suddendeath may also occur. For weaners and growers, pigs with glässersdisease become rapidly depressed or may be just found dead. Othersymptoms include elevated temperature, anorexia, a reluctance to rise,nervous signs such as fits and convulsions including meningitis, andpoor pigs, that are wasting and hairy often result. In young growingpigs, the following symptoms are most common: fever, mild meningitis,arthritis, lameness, pneumonia, heart sac infection, peritonitis andpleurisy. Again, a characteristic feature is a short cough of only 2-3episodes.

Exudative epidermitis is caused by the bacterium Staphylococcus hyicuswhich lives normally on the skin without causing disease. It is notknown why sometimes it flares up and causes a dermatitis which oozesgreasy fluid. It produces toxins which are absorbed into the system anddamage the liver and kidneys. In the sucking piglet disease is usuallyconfined to individual animals, but it can be a major problem in newgilt herds and weaned pigs. During the days immediately precedingfarrowing, the bacterium multiples profusely in the sow's vagina so thatpiglets are infected during the birth process or soon thereafter.Symptoms in sows include uncommon but localised lesions may be seenparticularly behind the face and eyes. Severely affected piglets willdie. In piglets, symptoms include localised lesions on the flanks andbehind ears. Lesions usually commence with small, dark, localised areasof infection around the face or on the legs. The skin along the flanksthe belly and between the legs changes to a brown color, graduallyinvolving the whole of the body. The skin becomes wrinkled with flakingof large areas and it has a greasy feel. In severe cases, the skin turnsblack due to necrosis and the piglets die. A more localised picture isseen if the sow has passed some immunity to the piglet, with smallcircumscribed lesions approximately 5-10 mm in diameter that do notspread. For weaners and growers, symptoms usually commence about 3 daysafter weaning with localised, brown areas of infection or dermatitisaround the face or on the legs, where the skin has been damaged. It mayulcerate. The skin along the flanks the belly and between the legschanges to a brown colour gradually involving the whole of the body. Theskin becomes wrinkled with flaking of large areas and progresses to adark greasy texture and in severe cases turns black. Such cases usuallydie due to the toxins produced by the staphylococci organisms. Innurseries up to 15% of the population may be involved and dehydration iscommon.

Swine erysipelas is caused by a bacterium, Erysipelothrix rhusiopathiaethat is found in most if not all pig farms. Up to 50% of animals maycarry it in their tonsils. It is always present in either the pig or inthe environment because it is excreted via saliva, feces or urine. It isalso found in many other species, including birds and sheep, and cansurvive outside the pig for a few weeks and longer in light soils. Thusit is impossible to eliminate it from a herd. Infected feces areprobably the main source of infection, particularly in growing andfinishing pens. The bacterium alone can cause the disease but concurrentvirus infections, such as PRRS or influenza, may trigger off outbreaks.Disease is relatively uncommon in pigs under 8-12 weeks of age due toprotection provided by maternal antibodies from the sow via thecolostrum. The most susceptible animals are growing pigs, non vaccinatedgilts, and up to 4th parity sows. The organism multiplies in the body,and invades the bloodstream to produce a septicaemia. The rapidity ofmultiplication and the level of immunity in the pig then determines theclinical symptoms.

Eperythrozoonosis (Epe) is a disease caused by a bacterium calledEperythrozoonosis suis which attaches to the surface of red blood cellsand sometimes destroys them. The pig may then become anaemic and theproducts left after the destruction of the cells may cause jaundice.Clinical disease is more commonly seen in young growing pigs. However itcan also cause reproductive problems in the breeding herd. A sow maycarry Epe and yet remain quite healthy, however, it can cross theplacenta resulting in weak pale pigs at birth. Epe is present in most ifnot all herds but the mechanisms which allow it to become pathogenic andproduce disease in some populations and not in others are unknown. Theincidence of disease is low.

Encephalomyocarditis, or EMC, infects and causes disease in a wide rangeof vertebrate animals but pigs appear to be the most susceptible of farmanimal species. The virus is world-wide but differs in pathogenicity andvirulence in different countries and regions. In most countries ofEurope, particularly those in the EU, it tends to be relatively mild ornon-pathogenic and disease in pigs is rarely diagnosed. In Australia thestrains appear to be much more virulent for pigs than those in NewZealand. Virulent strains in Florida, the Caribbean and probably CentralAmerica damage the heart and cause death whereas those in the Mid Westof the US tend to cause reproductive problems. Clinical disease in pigstends to occur when rat numbers increase to plague levels. Pigs can beinfected from rats or from rat-contaminated feed or water. It does notseem to spread very readily between pigs. In affected herds there areusually no clinical signs in weaned and growing pigs.

Aujeszky's disease, or AD, is an important disease of pigs caused by aherpes virus. The virus can remain hidden in nerves of the pig in acarrier state for long periods of time and then be reactivated. Onceintroduced into a herd, the virus usually remains there and it cancontinually affect reproductive performance at varying levels. The viruscan survive for up to three weeks outside the pig. Acute outbreaks ofdisease occur when virulent strains of the virus first infect anunvaccinated susceptible herd. The virus crosses the uterus and placentaand infects the foetuses. The pig is the main host. However, dogs andcattle may also become infected, show nervous signs, and die.

Porcine Cytomegalovirus Infection (PCMV) is caused by a herpes virusfound in the tissues throughout the body including the nose of newbornpiglets where it causes inflammation (rhinitis). PCMV is presentthroughout the world and exists in most if not all pig populations butmost infections are sub-clinical and clinical disease is rare. Serologycarried out in the UK, for example, indicates that over 90% of herdshave been exposed to infection. The rhinitis produced by this virus isuncommon and occurs mainly in newborn pigs and has no relationship toatrophic rhinitis caused by the toxin-producing bacteria Pasteurellamultocidia. In most herds therefore the infection is insignificant andapart from sometimes causing a mild sneeze has no major effect on thehealth of the pig.

Blue Eye Disease is a viral disease that causes nervous symptoms,reproductive failure and opacity or blueing of the cornea. It is seenmainly in Mexico but has also been reported in other countries. It isnot seen in Europe. Symptoms include inappetence, cornealopacity—conjunctivitis, nervous signs such as fits and convulsions, atendency to sit like a dog, fever, increased returns, increased weaningto mating intervals, stillbirths, mummified piglets, high mortality inpiglets, swollen testicles, and loss of libido.

Japanese B Encephalitis Virus (JE) is a virus spread by mosquitoes andis only important in countries where the insects are prevalent. Mostdomestic animals are affected. It causes an encephalitis in the human.The pig is an important source of infection. Symptoms include mummifiedor stillborn piglets, nervous signs in piglets such as fits andconvulsions, and oedema fluid in piglets. It can also cause infertilityand swollen testicles in boars.

Porcine Epidemic Diarrhoea (PED) is caused by a coronavirus somewhatsimilar to that which causes TGE. This virus is widespread in Europe.The virus damages the villi in the gut thus reducing the absorptivesurface, with loss of fluid and dehydration. After introduction of thevirus into a susceptible breeding herd, a strong immunity develops overtwo to three weeks. The colostral immunity then protects the piglets.The virus usually disappears spontaneously from breeding herdsparticularly small ones (<300 sows). Acute outbreaks of diarrhoea occurwhen the virus is first introduced into a susceptible population. Insuch cases up to 100% of sows may be affected, showing a mild to verywatery diarrhoea. Two clinical pictures are recognised: PED Type I onlyaffects growing pigs whereas PED Type II affects all ages includingsucking pigs and mature sows. The incubation period is approximately 2days and diarrhea lasts for 7 to 14 days. In sucking pigs, the diseasecan be mild or severe with mortalities up to 40%. In large breedingherds, particularly if kept extensively, not all the females may becomeinfected first time around and there may be recrudescence. This onlyoccurs in piglets suckling from sows with no maternal antibodies and istherefore sporadic.

Porcine Respiratory Corona Virus Infection (PRCV) first appeared in pigsin Europe some ten years or more ago. It is related to but distinct fromTGE virus, which is another corona virus. It is thought to spreadbetween farms on wind and so it is extremely difficult to keep herdsfree from it. Infection often takes place in the sucking pig at 2 to 3weeks of age but is not of importance. It may have an effect on lungtissue when other respiratory pathogens are present in chronicrespiratory disease complexes. Sows usually present no symptoms, butcoughing may occur in the presence of other respiratory agents coughingmay be associated. In piglets, a transient cough may be present. Inweaners and growers, herds exposed for the first time have few if anysigns of disease. The most common symptom is a transient coughinglasting only a few hours.

Rotavirus Infection is a virus infection that is widespread in pigpopulations. It is present in most if not all pig herds with virtually a100% sero-conversion in adult stock. A further epidemiological featureis its persistence outside the pig where it is resistant toenvironmental changes and many disinfectants. Maternal antibodiespersist for 3-6 weeks after which pigs become susceptible to infectionbut exposure does not necessarily result in disease. It is estimatedthat only 10-15% of diarrheas in pigs are initiated by a primaryrotavirus infection. In a mature herd, disease appears after piglets are7 to 10 days of age. It becomes progressively less important with age.However if pathogenic strains of E. coli are present, severe disease canoccur with heavy mortality.

Rabies is caused by a virus and considered a rare disease in pigs. It isinvariably fatal in all species including the human—hence itsimportance. Rabies is absent from the UK but present in may othercountries throughout the world. Infection in piglets and sows is rare.In sows, weaners, and growers, the onset of disease is sudden withsymptoms that include a nervous twitching of the face muscles, fits andconvulsions, rapid chewing, salivation, muscles that may go into spasm,and posterior paralysis may occur. Death usually takes place within 3days.

Swine Vesicular Disease (SVD) is a different virus from the virus thatcauses foot and mouth disease (FMD). However, it produces a disease inpigs that is clinically indistinguishable from FMD. This disease shouldalways be considered if sudden widespread lameness appears with vesiclesor blisters on the snout, tongue and tops of the claws. Tuberculosisaffects mammals, including people, birds, and swine. The causalorganism, Mycobacterium tuberculosis, is sub-classified into types,human, bovine and avian. The avian type is referred to as M. avium ormore often the avian/intracellulare complex because it is not a uniformspecies. M. avium itself infects mainly birds but is also found in theenvironment along with M. intracellulare which is predominantlysaprophytic or free living. Pigs are rarely infected by the human orbovine types but are commonly infected by the avian/intracellularecomplex. The avian/intracellulare complex also causes sub-clinicalnon-progressive infection in healthy people. The main concern is that itcould cause more serious disease in immuno-suppressed people and peoplewith AIDS. In most countries if lesions are found in the neck atslaughter the whole head is condemned and if they are found in themesenteric lymph nodes which drain the intestines the offals arecondemned. If they are more widespread in the body, which is rare, thewhole carcass may be condemned or cooked. If small lesions are missed bythe meat inspector normal kitchen cooking destroys the organism. In allpigs, infection causes small nodules in the lymph nodes of the neck andthose that drain the small intestine. In the great majority of cases thelesions are non-progressive, they do not spread through the body, do notmake the pig ill and are not excreted. There are no clinical symptomsand there is no difference in performance between infected andnon-infected pigs.

The virus of vesicular exanthema of swine (VES) is different from thosecausing foot-and-mouth disease (FMD) and swine vesicular disease (SVD)but it produces a disease in pigs that is clinically indistinguishablefrom FMD and SVD. Unlike FMD, it only effects pigs. Symptoms include lowmortality, but there may be some deaths in suckling piglets. Othersymptoms include salivation, inappetance, and vesicles around the mouth,nose, tongue and feet.

Vesicular Stomatitis (VS) causes a disease that occurs mainly in Southand Central America, occasionally in the USA and rarely as epidemicsextending as far North as Canada and as far South as Argentina. The VSvirus produces a disease in pigs that is clinically indistinguishableFMD, SVD and VES. Most often however infection of pigs is subclinical.In all pigs, infection is characterized by drooling saliva, foot lesionsand lameness, a reduction in growth rate, a rise in body temperature to40-41° C. (106-107° F.) the appearance of vesicles (blisters) up to 30mm diameter on the nose, lips, and teats and around the coronets of thefeet which may make the pigs lame. Mortality is usually low and mostpigs recover in one to two weeks.

Atrophic Rhinitis, Progressive and Unprogressive Disease which causesinflammation of the nose and it can be caused by a variety of bacteriaand irritant substances. During the process of infection, the delicatestructures or turbinate bones in the nose become damaged and atrophy ordisappear. Progressive atrophic rhinitis describes a specific diseasewhere the nose tissues permanently atrophy. It is caused by specifictoxin producing strains of Pasteurella multocidia (PMt). There are twotypes A and D. In sucking pigs, sneezing, snuffling and a nasaldischarge are the first symptoms, but in acute outbreaks where there islittle maternal antibody, the rhinitis may be so severe to the extentthat there is haemorrhage from the nose. By three to four weeks of ageand from weaning onwards, there is evidence of tear staining andmalformation of the nose associated with twisting and shortening.Severely affected pigs may have problems eating. There is considerablyreduced daily gain. In severe outbreaks pigs may not grow to marketweight.

Eastern equine encephalomyelitis viruses (EEEV) are members of theAlphavirus genus, family Togaviridae. EEEV can be transmitted to equinesand humans during the bite of an infected mosquito. In addition tohorses and humans, EEEV can produce severe disease in common livestockspecies such as swine and cattle. EEEV, or virus-specific antibodies,have been recovered from birds such as the turkey, pheasant, quail,ostrich, and emu, among others.

Mycoplasma arthritis is caused by Mycoplasma hyosynoviae infection. Thisarthritis, is characterized by inflammation of one or more joints and iscommon in all sucking and growing pigs and sows. However, it is rare inpiglets.

Infection in swine is also caused by adenovirus and hemagglutinatingencephalomyelitis virus.

Accordingly, what is needed in the art is a method of obtaining ORF2protein, which does not require extraction of the ORF2 protein fromwithin infected cells. What is further needed are methods of obtainingrecombinant ORF2 protein in quantities sufficient for efficientlypreparing vaccine compositions. What is still further needed are methodsfor obtaining ORF2 protein which do not require the complicated andlabor-intensive methods required by the current ORF2 protein extractionprotocols. Finally, with respect to compositions, what is needed in theart is an immunogenic composition which does confer protective immunityagainst PCV2 infection and lessens the severity of or prevents theclinical signs associated therewith.

SUMMARY OF THE INVENTION

The present invention overcomes the problems inherent in the prior artand provides a distinct advance in the state of the art. Specifically,one aspect of the present invention provides improved methods ofproducing and/or recovering recombinant PCV2 ORF2 protein, i) bypermitting infection of susceptible cells in culture with a recombinantviral vector containing PCV2 ORF2 DNA coding sequences, wherein ORF2protein is expressed by the recombinant viral vector, and ii) thereafterrecovering the ORF2 in the supernate. It has been unexpectedlydiscovered that ORF2 is released into the supernate in large quantitiesif the infection and subsequent incubation of the infected cells isallowed to progress past the typical prior PCV 2 ORF2 recovery process,which extracts the PCV2 ORF2 from within cells. It furthermore has beensurprisingly found, that PCV ORF2 protein is robust against prototypicaldegradation outside of the production cells. Both findings togetherallow a recovery of high amounts of PCV2 ORF2 protein from the supernateof cell cultures infected with recombinant viral vectors containing aPCV2 ORF2 DNA and expressing the PCV2 ORF2 protein. High amounts of PCV2ORF2 protein means more than about 20 μg/mL supernate, preferably morethan about 25 μg/mL, even more preferably more than about 30 μg/mL, evenmore preferably more than about 40 μg/mL, even more preferably more thanabout 50 μg/mL, even more preferably more than about 60 μg/mL, even morepreferably more than about 80 μg/mL, even more preferably more thanabout 100 μg/mL, even more preferably more than about 150 kg/mL, mostpreferably than about 190 μg/mL. Those expression rates can also beachieved for example by the methods as described in Examples 1 to 3.

Preferred cell cultures have a cell count between about 0.3-2.0×10⁶cells/mL, more preferably from about 0.35-1.9×10⁶ cells/mL, still morepreferably from about 0.4-1.8×10⁶ cells/mL, even more preferably fromabout 0.45-1.7×10⁶ cells/mL, and most preferably from about 0.5-1.5×10⁶cells/mL. Preferred cells are determinable by those of skill in the art.Preferred cells are those susceptible for infection with an appropriaterecombinant viral vector, containing a PCV2 ORF2 DNA and expressing thePCV2 ORF2 protein. Preferably the cells are insect cells, and morepreferably, they include the insect cells sold under the trademark Sf+insect cells (Protein Sciences Corporation, Meriden, Conn.).

Appropriate growth media will also be determinable by those of skill inthe art with a preferred growth media being serum-free insect cell mediasuch as Excell 420 (JRH Biosciences, Inc., Lenexa, Kans.) and the like.Preferred viral vectors include baculovirus such as BaculoGold (BDBiosciences Pharmingen, San Diego, Calif.), in particular if theproduction cells are insect cells. Although the baculovirus expressionsystem is preferred, it is understood by those of skill in the art thatother expression systems will work for purposes of the presentinvention, namely the expression of PCV2 ORF2 into the supernatant of acell culture. Such other expression systems may require the use of asignal sequence in order to cause ORF2 expression into the media. It hasbeen surprisingly discovered that when ORF2 is produced by a baculovirusexpression system, it does not require any signal sequence or furthermodification to cause expression of ORF2 into the media. It is believedthat this protein can independently form virus-like particles (Journalof General Virology Vol. 81, pp. 2281-2287 (2000) and be secreted intothe culture supernate. The recombinant viral vector containing the PCV2ORF2 DNA sequences has a preferred multiplicity of infection (MOI) ofbetween about 0.03-1.5, more preferably from about 0.05-1.3, still morepreferably from about 0.09-1.1, and most preferably from about 0.1-1.0,when used for the infection of the susceptible cells. Preferably theMOIs mentioned above relates to one mL of cell culture fluid.Preferably, the method described herein comprises the infection of0.35-1.9×10⁶ cells/mL, still more preferably of about 0.4-1.8×10⁶cells/mL, even more preferably of about 0.45-1.7×10⁶ cells/mL, and mostpreferably of about 0.5-1.5×10⁶ cells/mL with a recombinant viral vectorcontaining a PCV2 ORF2 DNA and expressing the PCV2 ORF protein having aMOI (multiplicity of infection) of between about 0.03-1.5, morepreferably from about 0.05-1.3, still more preferably from about0.09-1.1, and most preferably from about 0.1-1.0.

The infected cells are then incubated over a period of up to ten days,more preferably from about two days to about ten days, still morepreferably from about four days to about nine days, and most preferablyfrom about five days to about eight days. Preferred incubationconditions include a temperature between about 22-32° C., morepreferably from about 24-30° C., still more preferably from about 25-29°C., even more preferably from about 26-28° C., and most preferably about27° C. Preferably, the Sf+ cells are observed following inoculation forcharacteristic baculovirus-induced changes. Such observation may includemonitoring cell density trends and the decrease in viability during thepost-infection period. It was found that peak viral titer is observed3-5 days after infection and peak ORF2 release from the cells into thesupernate is obtained between days 5 and 8, and/or when cell viabilitydecreases to less than 10%.

Thus, one aspect of the present invention provides an improved method ofproducing and/or recovering recombinant PCV2 ORF2 protein, preferably inamounts described above, by i) permitting infection of a number ofsusceptible cells (see above) in culture with a recombinant viral vectorwith a MOI as defined above, ii) expressing PCV2 ORF2 protein by therecombinant viral vector, and iii) thereafter recovering the PCV2 ORF2in the supernate of cells obtained between days 5 and 8 after infectionand/or cell viability decreases to less then 10%. Preferably, therecombinant viral vector is a recombinant baculovirus containing PCV2ORF2 DNA coding sequences and the cells are Sf+ cells. Additionally, itis preferred that the culture be periodically examined for macroscopicand microscopic evidence of contamination or for atypical changes incell morphology during the post-infection period. Any culture exhibitingany contamination should be discarded. Preferably, the expressed ORF2recombinant protein is secreted by the cells into the surrounding growthmedia that maintains cell viability. The ORF2 is then recovered in thesupernate surrounding the cells rather than from the cells themselves.

The recovery process preferably begins with the separation of celldebris from the expressed ORF2 in media via a separation step. Preferredseparation steps include filtration, centrifugation at speeds up toabout 20,000×g, continuous flow centrifugation, chromatographicseparation using ion exchange or gel filtration, and conventionalimmunoaffinity methods. Those methods are known to persons skilled inthe art for example by (Harris and Angel (eds.), Protein purificationmethods—a practical approach, IRL press Oxford 1995). The most preferredseparation methods include centrifugation at speeds up to about 20,000×gand filtration. Preferred filtration methods include dead-endmicrofiltration and tangential flow (or cross flow) filtration includinghollow fiber filtration dead-end micro filtration. Of these, dead-endmicrofiltration is preferred. Preferred pore sizes for dead-endmicrofiltration are between about 0.30-1.35 μm, more preferably betweenabout 0.35-1.25 μm, still more preferably between about 0.40-1.10 μm,and most preferably between about 0.45-1.0 μm. It is believed that anyconventional filtration membrane will work for purposes of the presentinvention and polyethersulfone membranes are preferred. Any low weightnucleic acid species are removed during the filtration step.

Thus, one further aspect of the present invention provides an improvedmethod of producing and/or recovering recombinant PCV2 ORF2 protein,preferably in amounts described above, by i) permitting infection of anumber of susceptible cells (see above) in culture with a recombinantviral vector with a MOI as defined above, ii) expressing PCV ORF2protein by the recombinant viral vector, iii) recovering the PCV2 ORF2in the supernate of cells obtained between days 5 and 8 after infectionand/or cell viability decreases to less then 10%, and, iv) separatingcell debris from the expressed PCV2 ORF2 via a separation step.Preferably, the recombinant viral vector is a baculovirus containingORF2 DNA coding sequences and the cells are SF+ cells. Preferredseparation steps are those described above. Most preferred is a dead-endmicrofiltration using a membrane having a pore size between about0.30-1.35 μm, more preferably between about 0.35-1.25 μm, still morepreferably between about 0.40-1.10 μm, and most preferably between about0.45-1.0 μm.

For recovery of PCV2 ORF2 that will be used in an immunogenic orimmunological composition such as a vaccine, the inclusion of aninactivation step is preferred in order to inactivate the viral vector.An “immunogenic or immunological composition” refers to a composition ofmatter that comprises at least one antigen which elicits animmunological response in the host of a cellular and/orantibody-mediated immune response to the composition or vaccine ofinterest. Usually, an “immunological response” includes but is notlimited to one or more of the following effects: the production oractivation of antibodies, B cells, helper T cells, suppressor T cells,and/or cytotoxic T cells and/or yd T cells, directed specifically to anantigen or antigens included in the composition or vaccine of interest.Preferably, the host will display either a therapeutic or protectiveimmunological response such that resistance to new infection will beenhanced and/or the clinical severity of the disease reduced. Suchprotection will be demonstrated by either a reduction or lack ofsymptoms normally displayed by an infected host, a quicker recovery timeand/or a lowered viral titer in the infected host. Thus, the presentinvention also relates to method of producing and/or recoveringrecombinant PCV2 ORF2 protein, preferably in amounts described above, byi) permitting infection of a number of susceptible cells (see above) inculture with a recombinant viral vector with a MOI as defined above, ii)expressing PCV ORF2 protein by the recombinant viral vector, iii)recovering the PCV2 ORF2 in the supernate of cells obtained between days5 and 8 after infection and/or cell viability decreases to less then10%, iv) separating cell debris from the expressed PCV2 ORF2 via aseparation step, and v) inactivating the recombinant viral vector.

Preferably, this inactivation is done either just before or just afterthe filtration step, with after the filtration step being the preferredtime for inactivation. Any conventional inactivation method can be usedfor purposes of the present invention. Thus, inactivation can beperformed by chemical and/or physical treatments. In preferred forms,the volume of harvest fluids is determined and the temperature isbrought to between about 32-42° C., more preferably between about 34-40°C., and most preferably between about 35-39° C. Preferred inactivationmethods include the addition of cyclized binary ethylenimine (BEI),preferably in a concentration of about 1 to about 20 mM, more preferablyof about 2 to about 10 mM, still more preferably of about 2 to about 8mM, still more preferably of about 3 to about 7 mM, and most preferablyof about 5 mM. For example the inactivation includes the addition of asolution of 2-bromoethyleneamine hydrobromide, preferably of about 0.4M,which has been cyclized to 0.2M binary ethylenimine (BEI) in 0.3N NaOH,to the fluids to give a final concentration of about 5 mM BEI.Preferably, the fluids are then stirred continuously for 72-96 hours andthe inactivated harvest fluids can be stored frozen at −40° C. or belowor between about 1-7° C. After inactivation is completed, a sodiumthiosulfate solution, preferably at 1.0M is added to neutralize anyresidual BEI. Preferably, the sodium thiosulfate is added in equivalentamount as compared to the BEI added prior to for inactivation. Forexample, in the event BEI is added to a final concentration of 5 mM, a1.0M sodium thiosulfate solution is added to give a final minimumconcentration of 5 mM to neutralize any residual BEI.

Thus, one further aspect of the present invention relates to a method ofproducing recombinant PCV2 ORF2 protein, preferably in amounts describedabove, by i) permitting infection of a number of susceptible cells (seeabove) in culture with a recombinant viral vector with a MOI as definedabove, ii) expressing PCV ORF2 protein by the recombinant viral vector,iii) recovering the PCV2 ORF2 in the supernate of cells obtained betweendays 5 and 8 after infection and/or cell viability decreases to lessthen 10%, iv) separating cell debris from the expressed PCV2 ORF2 via aseparation step, and v) inactivating the recombinant viral vector.Preferably, the recombinant viral vector is a baculovirus containingORF2 DNA coding sequences and the cells are SF+ cells. Preferredseparation steps are those described above, most preferred is thefiltration step. Preferred inactivation steps are those described above.Preferably, inactivation is performed between about 35-39° C. and in thepresence of 2 to 8 mM BEI, and still more preferably in the presence ofabout 5 mM BEI. It has been surprisingly found, that higherconcentrations of BEI negatively affect the PCV2 ORF2 protein.

According to one further aspect of the present invention, the methoddescribed above also includes a neutralization step after step v). Thisstep vi) comprises adding of an equivalent amount of an agent thatneutralizes the inactivation agent within the solution. Preferably, ifthe inactivation agent is BEI, addition of sodium thiosulfate to anequivalent amount is preferred. Thus, according to a further aspect,step vi) comprises adding a sodium thiosulfate solution to a finalconcentration of about 1 to about 20 mM, preferably of about 2 to about10 mM, still more preferably of about 2 to about 8 mM, still morepreferably of about 3 to about 7 mM most preferably of about 5 mM, whenthe inactivation agent is BEI.

In preferred forms and especially in forms that will use the recombinantPCV2 ORF2 protein in an immunogenic composition such as a vaccine, eachlot of harvested ORF2 will be tested for inactivation by passage in theanchorage dependent, baculovirus susceptible Sf+ cells. In a preferredform of this testing, 150 cm² of appropriate cell culture monolayer isinoculated with 1.0 mL of inactivated PCV2 fluids and maintained at25-29° C. for 14 days with at least two passages. At the end of themaintenance period, the cell monolayers are examined for cytopathogeniceffect (CPE) typical of PCV2 ORF2 baculovirus. Preferably, positivevirus controls are also used. Such controls can consist of one cultureof Sf+ cells inoculated with a non-inactivated reference PCV2 ORF2baculovirus and one flask of Sf+ cells that remain uninoculated. Afterincubation and passage, the absence of virus-infected cells in the BEItreated viral fluids would constitute a satisfactory inactivation test.The control cells inoculated with the reference virus should exhibit CPEtypical of PCV2 ORF2 baculovirus and the uninoculated flask should notexhibit any evidence of PCV2 ORF2 baculovirus CPE. Alternatively, at theend of the maintenance period, the supernatant samples could becollected and inoculated onto a Sf+96 well plate, which has been loadedwith Sf+ cells, and then maintained at 25-29° C. for 5-6 days. The plateis then fixed and stained with anti-PCV2 ORF2 antibody conjugated toFITC. The absence of CPE and ORF2 expression, as detected by IFAmicoscopy, in the BEI treated viral fluids constitutes a satisfactoryinactivation test. The control cells inoculated with the reference virusshould exhibit CPE and IFA activity and the uninoculated flask shouldnot exhibit any evidence of PCV2 ORF2 baculovirus CPE and contain no IFAactivity.

Thus a further aspect of the present invention relates to aninactivation test for determining the effectiveness of the inactivationof the recombination viral vector, comprising the steps: i) contactingat least a portion of the culture fluid containing the recombinant viralvector with an inactivating agent, preferably as described above, ii)adding a neutralization agent to neutralize the inactivation agent,preferably as described above, and iii) determining the residualinfectivity by the assays as described above.

A further aspect of the invention relates to a method for constructing arecombinant viral vector containing PCV2 ORF2 DNA and expressing PCV2ORF2 protein in high amounts, when infected into susceptible cells. Ithas been surprisingly found that the recombinant viral vector asprovided herewith expresses high amounts, as defined above, of PCV2 ORF2after infecting susceptible cells. Therefore, the present invention alsorelates to an improved method for producing and/or recovering of PCV2ORF2 protein, preferably comprising the steps of: constructing arecombinant viral vector containing PCV2 ORF2 DNA and expressing PCV2ORF2 protein. Preferably, the viral vector is a recombinantbaculorvirus. Details of the method for constructing recombinant viralvectors containing PCV2 ORF2 DNA and expressing PCV2 ORF2 protein, asprovided herewith, are described to the following: In preferred formsthe recombinant viral vector containing PCV2 ORF 2 DNA and expressingPCV2 ORF2 protein used to infect the cells is generated by transfectinga transfer vector that has had an ORF2 gene cloned therein into a viralvector. Preferably, only the portion of the transfer vector, thatcontains the ORF2 DNA is transfected into the viral vector. The term“transfected into a viral vector” means, and is used as a synonym for“introducing” or “cloning” a heterologous DNA into a viral vector, suchas for example into a baculovirus vector. The viral vector is preferablybut not necessarily a baculovirus.

Thus, according to a further aspect of the present invention, therecombinant viral vector is generated by recombination between atransfer vector containing the heterologous PCV2 ORF2 DNA and a viralvector, preferably a baculorvirus, even more preferably a linearizedreplication-deficient baculovirus (such as Baculo Gold DNA). A “transfervector” means a DNA molecule, that includes at least one origin ofreplication, the heterologous gene, in the present case PCV2 ORF2, andDNA sequences which allow the cloning of said heterologous gene into theviral vector. Preferably the sequences which allow cloning of theheterologous gene into the viral vector are flanking the heterologousgene. Even more preferably, those flanking sequences are at leasthomologous in parts with sequences of the viral vector. The sequencehomology then allows recombination of both molecules, the viral vector,and the transfer vector to generate a recombinant viral vectorcontaining the heterologous gene. One preferred transfer vector is thepVL1392 vector (BD Biosciences Pharmingen), which is designed forco-transfection with the BaculoGold DNA into the preferred Sf+ cellline. Preferably, said transfer vector comprises a PCV2 ORF2 DNA. Theconstruct co-transfected is approximately 10,387 base pairs in length.

In more preferred forms, the methods of the present invention will beginwith the isolation of PCV2 ORF2 DNA. Generally, this can be from a knownor unknown strain as the ORF2 DNA appears to be highly conserved with atleast about 95% sequence identity between different isolates. Any PCV2ORF2 gene known in the art can be used for purposes of the presentinvention as each would be expressed into the supernate. The PCV ORF2DNA is preferably amplified using PCR methods, even more preferablytogether with the introduction of a 5′ flanking Kozak's consensussequence (CCGCCAUG) (SEQ ID NO 1) and/or a 3′ flanking EcoR1 site(GAATTC) (SEQ ID NO 2). Such introduction of a 5′ Kozak's consensuspreferably removes the naturally occurring start codon AUG of PCV2 ORF2.The 3′ EcoR1 site is preferably introduced downstream of the stop codonof the PCV2 ORF2. More preferably it is introduced downstream of a polyA transcription termination sequence, that itself is located downstreamof the PCV2 ORF2 stop codon. It has been found, that the use of aKozak's consensus sequence, in particular as described above, increasesthe expression level of the subsequent PCV2 ORF2 protein. The amplifiedPCV2 ORF2 DNA, with these additional sequences, is cloned into a vector.A preferred vector for this initial cloning step is the pGEM-T-EasyVector (Promega, Madison, Wis.). The PCV2 ORF2 DNA including some pGEMvector sequences (SEQ ID NO: 7) is preferably excised from the vector atthe Not1 restriction site. The resulting DNA is then cloned into thetransfer vector.

Thus, in one aspect of the present invention, a method for constructinga recombinant viral vector containing PCV2 ORF2 DNA is provided. Thismethod comprises the steps: i) cloning a recombinant PCV2 ORF2 into atransfer vector; and ii) transfecting the portion of the transfer vectorcontaining the recombinant PCV2 ORF2 into a viral vector, to generatethe recombinant viral vector. Preferably, the transfer vector is thatdescribed above or is constructed as described above or as exemplarilyshown in FIG. 1. Thus according to a further aspect, the transfervector, used for the construction of the recombinant viral vector asdescribed herein, contains the sequence of SEQ ID NO: 7.

According to a further aspect, this method further comprises prior tostep i) the following step: amplifying the PCV2 ORF2 DNA in vitro,wherein the flanking sequences of the PCV2 ORF2 DNA are modified asdescribed above. In vitro methods for amplifying the PCV2 ORF2 DNA andmodifying the flanking sequences, cloning in vitro amplified PCV2 ORF2DNA into a transfer vector and suitable transfer vectors are describedabove, exemplarily shown in FIG. 1, or known to a person skilled in theart. Thus according to a further aspect, the present invention relatesto a method for constructing a recombinant viral vector containing PCV2ORF2 DNA and expressing PCV2 ORF2 protein comprises the steps of: i)amplifying PCV2 ORF2 DNA in vitro, wherein the flanking sequences ofsaid PCV2 ORF2 DNA are modified, ii) cloning the amplified PCV2 ORF2 DNAinto a transfer vector; and iii) transfecting the transfer vector or aportion thereof containing the recombinant PCV2 ORF2 DNA into a viralvector to generate the recombinant viral vector. Preferably, themodification of the flanking sequences of the PCV2 ORF2 DNA is performedas described above, e.g. by introducing a 5′ Kozak's sequence and/or anEcoR 1 site, preferably as described above.

According to a further aspect, a method of producing and/or recoveringrecombinant protein expressed by open reading frame 2 of PCV2 isprovided. The method generally comprises the steps of: i) cloning arecombinant PCV2 ORF2 into a transfer vector; ii) transfecting theportion of the transfer vector containing the recombinant PCV2 ORF2 intoa virus; iii) infecting cells in media with the transfected virus; iv)causing the transfected virus to express the recombinant protein fromPCV2 ORF2; v) separating cells from the supernate; and vi) recoveringthe expressed PCV2 ORF2protein from the supernate.

Methods of how to clone a recombinant PCV2 ORF2 DNA into a transfervector are described above. Preferably, the transfer vector contains thesequence of SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:7. However, thetransfer vector can contain any PCV2 ORF2 DNA, unmodified or modified,as long as the PCV2 ORF2 DNA, when transfected into a recombinant viralvector, is expressed in cell culture. Preferably, the recombinant viralvector comprises the sequence of SEQ ID NO:8. Moreover, methods of howto infect cells, preferably how to infect insect cells with a definednumber of recombinant baculovirus containing PCV2 ORF2 DNA andexpressing PCV2 ORF2 protein, are described above in detail. Moreover,steps of separating cells from the supernate as well as steps forrecovering the expressed PCV2 ORF2 protein are also described above indetail. Any of these specific process steps, as described herein, arepart of the method of producing and/or recovering recombinant proteinexpressed by open reading frame 2 of PCV2 as described above.Preferably, the cells are SF+ cells. Still more preferably, cellcultures have a cell count between about 0.3-2.0×10⁶ cells/mL, morepreferably from about 0.35-1.9×10⁶ cells/mL, still more preferably fromabout 0.4-1.8×10⁶ cells/mL, even more preferably from about 0.45-1.7×10⁶cells/mL, and most preferably from about 0.5-1.5×10⁶ cells/mL.Preferably, the recombinant viral vector containing the PCV2 ORF2 DNAhas a preferred multiplicity of infection (MOI) of between about0.03-1.5, more preferably from about 0.05-1.3, still more preferablyfrom about 0.09-1.1, still more preferably from about 0.1-1.0, and mostpreferably to about 0.5, when used for the infection of the susceptiblecells. Preferably, recovering of the PCV2 ORF2 protein in the supernateof cells obtained between days 5 and 8 after infection and/or cellviability decreases to less then 10%. Preferably, for producing PCV2ORF2 protein, cells are cultivated at 25 to 29° C. Preferably, theseparation step is a centrifugation or a filtration step.

Optionally, this method can include the step of amplifying the PCV2 ORF2DNA from a strain of PCV2 prior to cloning the PCV2 ORF2 DNA into thetransfer vector. In preferred forms, a 5′ Kozak's sequence, a 3′ EcoR1site, and combinations thereof can also be added to the amplifiedsequence, preferably prior to or during amplification. A preferred 5′Kozak's sequence comprises SEQ ID NO: 1. A preferred 3′ EcoR1 sitecomprises SEQ ID NO: 2. Preferred PCV2 ORF2 DNA comprises the nucleotidesequence Genbank Accession No. AF086834 (SEQ ID NO: 3) and SEQ ID NO: 4.Preferred recombinant PCV2 ORF2 protein comprises the amino acidsequence of SEQ ID NO: 5, which is the protein encoded by SEQ ID NO: 3(Genbank Accession No. AF086834) and SEQ ID No: 6, which is the proteinencoded by SEQ ID NO: 4. A preferred media comprises serum-free insectcell media, still more preferably Excel 420 media. When the optionalamplification step is performed, it is preferable to first clone theamplified open reading frame 2 into a first vector, excise the openreading frame 2 from the first vector, and use the excised open readingframe for cloning into the transfer vector. A preferred cell line forcotransfection is the SF+ cell line. A preferred virus forcotransfection is baculovirus. In preferred forms of this method, thetransfected portion of the transfer vector comprises SEQ ID NO: 8.Finally, for this method, it is preferred to recover the PCV2 openreading frame 2 (ORF2) protein in the cell culture supernate at least 5days after infecting the cells with the virus.

Thus, a further aspect of the invention relates to a method forproducing and/or recovering the PCV2 open reading frame 2, comprises thesteps: i) amplifying the PCV2 ORF2 DNA in vitro, preferably by adding a5′ Kozak's sequence and/or by adding a 3′ EcoR1 restriction site, ii)cloning the amplified PCV2 ORF2 into a transfer vector; iii)transfecting the portion of the transfer vector containing therecombinant PCV2 ORF2 into a virus; iv) infecting cells in media withthe transfected virus; v) causing the transfected virus to express therecombinant protein from PCV2 ORF2; vi) separating cells from thesupernate; and vii) recovering the expressed PCV2 ORF2 protein from thesupernate.

A further aspect of the present invention relates to a method forpreparing a composition comprising PCV2 ORF2 protein, and inactivatedviral vector. This method comprises the steps: i) cloning the amplifiedPCV2 ORF2 into a transfer vector; ii) transfecting the portion of thetransfer vector containing the recombinant PCV2 ORF2 into a virus; iii)infecting cells in media with the transfected viral vector; iv) causingthe transfected viral vector to express the recombinant protein fromPCV2 ORF2; v) separating cells from the supernate; vi) recovering theexpressed PCV2 ORF2 protein from the supernate; and vii) inactivatingthe recombinant viral vector. Preferably, the recombinant viral vectoris a baculovirus containing ORF2 DNA coding sequences and the cells areSF+ cells. Preferred separation steps are those described above, mostpreferred is the filtration step. Preferred inactivation steps are thosedescribed above. Preferably, inactivation is performed between about35-39° C. and in the presence of 2 to 8 mM BEI, still more preferably inthe presence of about 5 mM BEI. It has been surprisingly found, thathigher concentrations of BEI negatively affect the PCV2 ORF2 protein,and lower concentrations are not effective to inactivate the viralvector within 24 to 72 hours of inactivation. Preferably, inactivationis performed for at least 24 hours, even more preferably for 24 to 72hours.

According to a further aspect, the method for preparing a compositioncomprising PCV2 ORF2 protein, and inactivated viral vector, as describedabove, also includes a neutralization step after step vii). This stepviii) comprises adding an equivalent amount of an agent that neutralizesthe inactivation agent within the solution. Preferably, if theinactivation agent is BEI, the addition of sodium thiosulfate to anequivalent amount is preferred. Thus, according to a further aspect,step viii) comprises adding a sodium thiosulfate solution to a finalconcentration of about 1 to about 20 mM, preferably of about 2 to about10 mM, still more preferably of about 2 to about 8 mM, still morepreferably of about 3 to about 7 mM, most preferably of about 5 mM, whenthe inactivation agent is BEI.

According to a further aspect, the method for preparing a compositioncomprising PCV2 ORF2 protein, and inactivated viral vector, as describedabove, comprises prior to step i) the following step: amplifying thePCV2 ORF2 DNA in vitro, wherein the flanking sequences of the PCV2 ORF2DNA are modified as described above. In vitro methods for amplifying thePCV2 ORF2 DNA and modifying the flanking sequences, cloning in vitroamplified PCV2 ORF2 DNA into a transfer vector and suitable transfervectors are described above, exemplarily shown in FIG. 1, or known to aperson skilled in the art. Thus according to a further aspect, thismethod comprises the steps: i) amplifying PCV2 ORF2 DNA in vitro,wherein the flanking sequences of said PCV2 ORF2 DNA are modified, ii)cloning the amplified PCV2 ORF2 DNA into a transfer vector; and iii)transfecting the transfer vector or a portion thereof containing therecombinant PCV2 ORF2 DNA into a viral vector to generate therecombinant viral vector, iv) infecting cells in media with thetransfected virus; v) causing the transfected virus to express therecombinant protein from PCV2 ORF2; vi) separating cells from thesupernate; vii) recovering the expressed PCV2 ORF2 protein from thesupernate; viii) inactivating the recombinant viral vector, preferably,in the presence of about 1 to about 20 mM BEI, most preferably in thepresence of about 5 mM BEI; and ix) adding an equivalent amount of anagent that neutralizes the inactivation agent within the solution,preferably, adding of a sodium thiosulfate solution to a finalconcentration of about 1 to about 20 mM, preferably of about 5 mM, whenthe inactivation agent is BEI.

In another aspect of the present invention, a method for preparing acomposition, preferably an antigenic composition, such as for example avaccine, for invoking an immune response against PCV2 is provided.Generally, this method includes the steps of transfecting a constructinto a virus, wherein the construct comprises i) recombinant DNA fromORF2 of PCV2, ii) infecting cells in growth media with the transfectedvirus, iii) causing the virus to express the recombinant protein fromPCV2 ORF2, iv) recovering the expressed ORF2 protein from the supernate,v) and preparing the composition by combining the recovered protein witha suitable adjuvant and/or other pharmaceutically acceptable carrier.

“Adjuvants” as used herein, can include aluminum hydroxide and aluminumphosphate, saponins e.g., Quil A, QS-21 (Cambridge Biotech Inc.,Cambridge Mass.), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham,Ala.), water-in-oil emulsion, oil-in-water emulsion,water-in-oil-in-water emulsion. The emulsion can be based in particularon light liquid paraffin oil (European Pharmacopea type); isoprenoid oilsuch as squalane or squalene oil resulting from theoligomerization ofalkenes, in particular of isobutene or decene; esters of acids or ofalcohols containing a linear alkyl group, more particularly plant oils,ethyl oleate, propylene glycol di-(caprylate/caprate), glyceryltri-(caprylate/caprate) or propylene glycol dioleate; esters of branchedfatty acids or alcohols, in particular isostearic acid esters. The oilis used in combination with emulsifiers to form the emulsion. Theemulsifiers are preferably nonionic surfactants, in particular esters ofsorbitan, of mannide (e.g. anhydromannitol oleate), of glycol, ofpolyglycerol, of propylene glycol and of oleic, isostearic, ricinoleicor hydroxystearic acid, which are optionally ethoxylated, andpolyoxypropylene-polyoxyethylene copolymer blocks, in particular thePluronic products, especially L121. See Hunter et al., The Theory andPractical Application of Adjuvants (Ed.Stewart-Tull, D. E. S.).JohnWiley and Sons, NY, pp51-94 (1995) and Todd et al., Vaccine15:564-570 (1997).

For example, it is possible to use the SPT emulsion described on page147 of “Vaccine Design, The Subunit and Adjuvant Approach” edited by M.Powell and M. Newman, Plenum Press, 1995, and the emulsion MF59described on page 183 of this same book.

A further instance of an adjuvant is a compound chosen from the polymersof acrylic or methacrylic acid and the copolymers of maleic anhydrideand alkenyl derivative. Advantageous adjuvant compounds are the polymersof acrylic or methacrylic acid which are cross-linked, especially withpolyalkenyl ethers of sugars or polyalcohols. These compounds are knownby the term carbomer (Phameuropa Vol. 8, No. 2, June 1996). Personsskilled in the art can also refer to U.S. Pat. No. 2,909,462 whichdescribes such acrylic polymers cross-linked with a polyhydroxylatedcompound having at least 3 hydroxyl groups, preferably not more than 8,the hydrogen atoms of at least three hydroxyls being replaced byunsaturated aliphatic radicals having at least 2 carbon atoms. Thepreferred radicals are those containing from 2 to 4 carbon atoms, e.g.vinyls, allyls and other ethylenically unsaturated groups. Theunsaturated radicals may themselves contain other substituents, such asmethyl. The products sold under the name Carbopol; (BF Goodrich, Ohio,USA) are particularly appropriate. They are cross-linked with an allylsucrose or with allyl pentaerythritol. Among then, there may bementioned Carbopol 974P, 934P and 971P. Most preferred is the use ofCabopol 971P. Among the copolymers of maleic anhydride and alkenylderivative, the copolymers EMA (Monsanto) which are copolymers of maleicanhydride and ethylene. The dissolution of these polymers in water leadsto an acid solution that will be neutralized, preferably tophysiological pH, in order to give the adjuvant solution into which theimmunogenic, immunological or vaccine composition itself will beincorporated.

Further suitable adjuvants include, but are not limited to, the RIBIadjuvant system (Ribi Inc.), Block co-polymer (CytRx, Atlanta Ga.),SAF-M (Chiron, Emeryville Calif.), monophosphoryl lipid A, Avridinelipid-amine adjuvant, heat-labile enterotoxin from E. coli (recombinantor otherwise), cholera toxin, IMS 1314 or muramyl dipeptide among manyothers.

Preferably, the adjuvant is added in an amount of about 100 μg to about10 mg per dose. Even more preferably, the adjuvant is added in an amountof about 100 μg to about 10 mg per dose. Even more preferably, theadjuvant is added in an amount of about 500 μg to about 5 mg per dose.Even more preferably, the adjuvant is added in an amount of about 750 μgto about 2.5 mg per dose. Most preferably, the adjuvant is added in anamount of about 1 mg per dose.

Thus, according to a further aspect, the method for preparing anantigenic composition, such as for example a vaccine, for invoking animmune response against PCV2 comprises i) preparing and recovering PCV2ORF2 protein, and ii) admixing this with a suitable adjuvant.Preferably, the adjuvant is Carbopol 971P. Even more preferably,Carbopol 971P is added in an amount of about 500 μg to about 5 mg perdose, even more preferably in an amount of about 750 μg to about 2.5 mgper dose and most preferably in an amount of about 1 mg per dose.Preferably, the process step i) includes the process steps as describedfor the preparation and recovery of PCV2 ORF2. For example, in preferredforms of this method, the construct comprising PCV2 ORF2 DNA is obtainedin a transfer vector. Suitable transfer vectors and methods of preparingthem are described above. Optionally, the method may include the step ofamplifying the ORF2 from a strain of PCV2 through PCR prior to cloningthe ORF2 into the transfer vector. Preferred open reading framesequences, Kozak's sequences, 3′ EcoR1 site sequences, recombinantprotein sequences, transfected construct sequences, media, cells, andviruses are as described in the previous methods. Another optional stepfor this method includes cloning the amplified PCV2 ORF2 DNA into afirst vector, excising the ORF2 DNA from this first vector, and usingthis excised PCV2 ORF2 DNA for cloning into the transfer vector. As withthe other methods, it is preferred to wait for at least 5 days afterinfection of the cells by the transfected baculovirus prior to recoveryof recombinant ORF2 protein from the supernate. Preferably, the recoverystep of this method also includes the step of separating the media fromthe cells and cell debris. This can be done in a variety of ways but forease and convenience, it is preferred to filter the cells, cell debris,and growth media through a filter having pores ranging in size fromabout 0.45 μM to about 1.0 μM. Finally, for this method, it is preferredto include a virus inactivation step prior to combining the recoveredrecombinant PCV2 ORF2 protein in a composition. This can be done in avariety of ways, but it is preferred in the practice of the presentinvention to use BEI.

Thus according to a further aspect, this method comprises the steps: i)amplifying PCV2 ORF2 DNA in vitro, wherein the flanking sequences ofsaid PCV2 ORF2 DNA are modified, ii) cloning the amplified PCV2 ORF2 DNAinto a transfer vector; and iii) transfecting the transfer vector or aportion thereof containing the recombinant PCV2 ORF2 DNA into a viralvector to generate the recombinant viral vector, iv) infecting cells inmedia with the transfected virus; v) causing the transfected virus toexpress the recombinant protein from PCV2 ORF2; vi) separating cellsfrom the supernate; vii) recovering the expressed PCV2 ORF2 protein fromthe supernate; viii) inactivating the recombinant viral vector,preferably, in the presence of about 1 to about 20 mM BEI, mostpreferably in the presence of about 5 mM BEI; ix) adding of anequivalent amount of an agent that neutralizes the inactivation agentwithin the solution, preferably, adding of a sodium thiosulfate solutionto a final concentration of about 1 to about 20 mM, preferably of about5 mM, when the inactivation agent is BEI, and x) adding a suitableamount of an adjuvant, preferably adding Carbopol, more preferablyCarbopol 971P, even more preferably in amounts as described above (e.g.of about 500 μg to about 5 mg per dose, even more preferably in anamount of about 750 μg to about 2.5 mg per dose and most preferably inan amount of about 1 mg per dose).

Additionally, the composition can include one or morepharmaceutical-acceptable carriers. As used herein, “apharmaceutical-acceptable carrier” includes any and all solvents,dispersion media, coatings, stabilizing agents, diluents, preservatives,antibacterial and antifungal agents, isotonic agents, adsorptiondelaying agents, and the like. Most preferably, the composition providedherewith, contains PCV2 ORF2 protein recovered from the supernate of invitro cultured cells, wherein said cells were infected with arecombinant viral vector containing PCV2 ORF2 DNA and expressing PCV2ORF2 protein, and wherein said cell culture was treated with about 2 toabout 8 mM BEI, preferably with about 5 mM BEI to inactivate the viralvector, and an equivalent concentration of a neutralization agent,preferably sodium thiosulfate solution, to a final concentration ofabout 2 to about 8 mM, preferably of about 5 mM, Carbopol, morepreferably Carbopol 971P, preferably in amounts of about 500 μg to about5 mg per dose, even more preferably in an amount of about 750 μg toabout 2.5 mg per dose and most preferably in an amount of about 1 mg perdose, and physiological saline, preferably in an amount of about 50 toabout 90% (v/v), more preferably to about 60 to 80% (v/v), still morepreferably of about 70% (v/v).

Thus, a further aspect relates to a method for preparing an antigeniccomposition, such as for example a vaccine, for invoking an immuneresponse against PCV2 comprising the steps: i) amplifying PCV2 ORF2 DNAin vitro, wherein the flanking sequences of said PCV2 ORF2 DNA aremodified, ii) cloning the amplified PCV2 ORF2 DNA into a transfervector; and iii) transfecting the transfer vector or a portion thereofcontaining the recombinant PCV2 ORF2 DNA into a viral vector to generatethe recombinant viral vector, iv) infecting cells in media with thetransfected virus; v) causing the transfected virus to express therecombinant protein from PCV2 ORF2; vi) separating cells from thesupernate; vii) recovering the expressed PCV2 ORF2 protein from thesupernate; viii) inactivating the recombinant viral vector, preferably,in the presence of about 2 to about 20 mM BEI, most preferably in thepresence of about 5 mM BEI; ix) adding an equivalent amount of an agentthat neutralize the inactivation agent within the solution, preferably,adding a sodium thiosulfate solution to a final concentration of about0.5 to about 20 mM, preferably of about 5 mM, when the inactivationagent is BEI, x) adding a suitable amount of an adjuvant, preferablyadding Carbopol, more preferably Carbopol 971P, still more preferably inamounts as described above (e.g. of about 500 μg to about 5 mg per dose,even more preferably in an amount of about 750 μg to about 2.5 mg perdose and most preferably in an amount of about 1 mg per dose); and xi)adding physiological saline, preferably in an amount of about 50 toabout 90% (v/v), more preferably to about 60 to 80% (v/v), still morepreferably of about 70% (v/v). Optionally, this method can also includethe addition of a protectant. A protectant as used herein, refers to ananti-microbiological active agent, such as for example Gentamycin,Merthiolate, and the like. In particular adding a protectant is mostpreferred for the preparation of a multi-dose composition. Thoseanti-microbiological active agents are added in concentrations effectiveto prevent the composition of interest from any microbiologicalcontamination or for inhibition of any microbiological growth within thecomposition of interest.

Moreover, this method can also comprise the addition of any stabilizingagent, such as for example saccharides, trehalose, mannitol, saccharoseand the like, to increase and/or maintain product shelf-life. However,it has been surprisingly found, that the resulting formulation isimmunologically effective over a period of at least 24 months, withoutadding any further stabilizing agent.

A further aspect of the present invention relates to the productsresulting from the methods as described above. In particular, thepresent invention relates to a composition of matter comprisingrecombinantly expressed PCV2 ORF2 protein. Moreover, the presentinvention also relates to a composition of matter that comprisesrecombinantly expressed PCV2 ORF2 protein, recovered from the supernateof an insect cell culture. Moreover, the present invention also relatesto a composition of matter comprising recombinantly expressed PCV2 ORF2protein, recovered from the supernate of an insect cell culture.Preferably, this composition of matter also comprises an agent suitablefor the inactivation of viral vectors. Preferably, said inactivationagent is BEI. Moreover, the present invention also relates to acomposition of matter that comprises recombinantly expressed PCV2 ORF2protein, recovered from the supernate of an insect cell culture, andcomprises an agent, suitable for the inactivation of viral vectors,preferably BEI and a neutralization agent for neutralization of theinactivation agent. Preferably, that neutralization agent is sodiumthiosulfate, when BEI is used as an inactivation agent.

In yet another aspect of the present invention, an immunogeniccomposition that induces an immune response and, more preferably,confers protective immunity against the clinical signs of PCV2infection, is provided. The composition generally comprises thepolypeptide, or a fragment thereof, expressed by Open Reading Frame 2(ORF2) of PCV2, as the antigenic component of the composition.

PCV2 ORF2 DNA and protein, as used herein for the preparation of thecompositions and also as used within the processes provided herein is ahighly conserved domain within PCV2 isolates and thereby, any PCV2 ORF2would be effective as the source of the PCV ORF2 DNA and/or polypeptideas used herein. A preferred PCV2 ORF2 protein is that of SEQ ID NO. 11.A preferred PCV ORF2 polypeptide is provided herein as SEQ ID NO. 5, butit is understood by those of skill in the art that this sequence couldvary by as much as 6-10% in sequence homology and still retain theantigenic characteristics that render it useful in immunogeniccompositions. The antigenic characteristics of an immunologicalcomposition can be, for example, estimated by the challenge experimentas provided by Example 4. Moreover, the antigenic characteristic of anmodified antigen is still retained, when the modified antigen confers atleast 70%, preferably 80%, more preferably 90% of the protectiveimmunity as compared to the PCV2 ORF 2 protein, encoded by thepolynucleotide sequence of SEQ ID NO:3 or SEQ ID NO:4. An “immunogeniccomposition” as used herein, means a PCV2 ORF2 protein which elicits an“immunological response” in the host of a cellular and/orantibody-mediated immune response to PCV2 ORF2 protein. Preferably, thisimmunogenic composition is capable of conferring protective immunityagainst PCV2 infection and the clinical signs associated therewith. Insome forms, immunogenic portions of PCV2 ORF2 protein are used as theantigenic component in the composition. The term “immunogenic portion”as used herein refers to truncated and/or substituted forms, orfragments of PCV2 ORF2 protein and/or polynucleotide, respectively.Preferably, such truncated and/or substituted forms, or fragments willcomprise at least 6 contiguous amino acids from the full-length ORF2polypeptide. More preferably, the truncated or substituted forms, orfragments will have at least 10, more preferably at least 15, and stillmore preferably at least 19 contiguous amino acids from the full-lengthORF2 polypeptide. Two preferred sequences in this respect are providedherein as SEQ ID NOs. 9 and 10. It is further understood that suchsequences may be a part of larger fragments or truncated forms. Afurther preferred PCV2 ORF2 polypeptide provided herein is encoded bythe nucleotide sequences of SEQ ID NO: 3 or SEQ ID NO: 4. But it isunderstood by those of skill in the art that this sequence could vary byas much as 6-20% in sequence homology and still retain the antigeniccharacteristics that render it useful in immunogenic compositions. Insome forms, a truncated or substituted form, or fragment of ORF2 is usedas the antigenic component in the composition. Preferably, suchtruncated or substituted forms, or fragments will comprise at least 18contiguous nucleotides from the full-length ORF2 nucleotide sequence,e.g. of SEQ ID NO: 3 or SEQ ID NO: 4. More preferably, the truncated orsubstituted forms, or fragments will have at least 30, more preferablyat least 45, and still more preferably at least 57 contiguousnucleotides the full-length ORF2 nucleotide sequence, e.g. of SEQ ID NO:3 or SEQ ID NO: 4.

“Sequence Identity” as it is known in the art refers to a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, namely a reference sequence and a given sequence to becompared with the reference sequence. Sequence identity is determined bycomparing the given sequence to the reference sequence after thesequences have been optimally aligned to produce the highest degree ofsequence similarity, as determined by the match between strings of suchsequences. Upon such alignment, sequence identity is ascertained on aposition-by-position basis, e.g., the sequences are “identical” at aparticular position if at that position, the nucleotides or amino acidresidues are identical. The total number of such position identities isthen divided by the total number of nucleotides or residues in thereference sequence to give % sequence identity. Sequence identity can bereadily calculated by known methods, including but not limited to, thosedescribed in Computational Molecular Biology, Lesk, A. N., ed., OxfordUniversity Press, New York (1988), Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York (1993); ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey (1994); Sequence Analysis in MolecularBiology, von Heinge, G., Academic Press (1987); Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York(1991); and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073(1988), the teachings of which are incorporated herein by reference.Preferred methods to determine the sequence identity are designed togive the largest match between the sequences tested. Methods todetermine sequence identity are codified in publicly available computerprograms which determine sequence identity between given sequences.Examples of such programs include, but are not limited to, the GCGprogram package (Devereux, J., et al., Nucleic Acids Research, 12(1):387(1984)), BLASTP, BLASTN and FASTA (Altschul, S. F. et al., J. Molec.Biol., 215:403-410 (1990). The BLASTX program is publicly available fromNCBI and other sources (BLAST Manual, Altschul, S. et al., NCVI NLM NIHBethesda, Md. 20894, Altschul, S. F. et al., J. Molec. Biol.,215:403-410 (1990), the teachings of which are incorporated herein byreference). These programs optimally align sequences using default gapweights in order to produce the highest level of sequence identitybetween the given and reference sequences. As an illustration, by apolynucleotide having a nucleotide sequence having at least, forexample, 85%, preferably 90%, even more preferably 95% “sequenceidentity” to a reference nucleotide sequence, it is intended that thenucleotide sequence of the given polynucleotide is identical to thereference sequence except that the given polynucleotide sequence mayinclude up to 15, preferably up to 10, even more preferably up to 5point mutations per each 100 nucleotides of the reference nucleotidesequence. In other words, in a polynucleotide having a nucleotidesequence having at least 85%, preferably 90%, even more preferably 95%identity relative to the reference nucleotide sequence, up to 15%,preferably 10%, even more preferably 5% of the nucleotides in thereference sequence may be deleted or substituted with anothernucleotide, or a number of nucleotides up to 15%, preferably 10%, evenmore preferably 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. These mutations of thereference sequence may occur at the 5′ or 3′ terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence. Analogously, by a polypeptide having a given aminoacid sequence having at least, for example, 85%, preferably 90%, evenmore preferably 95% sequence identity to a reference amino acidsequence, it is intended that the given amino acid sequence of thepolypeptide is identical to the reference sequence except that the givenpolypeptide sequence may include up to 15, preferably up to 10, evenmore preferably up to 5 amino acid alterations per each 100 amino acidsof the reference amino acid sequence. In other words, to obtain a givenpolypeptide sequence having at least 85%, preferably 90%, even morepreferably 95% sequence identity with a reference amino acid sequence,up to 15%, preferably up to 10%, even more preferably up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to15%, preferably up to 10%, even more preferably up to 5% of the totalnumber of amino acid residues in the reference sequence may be insertedinto the reference sequence. These alterations of the reference sequencemay occur at the amino or the carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in the one or more contiguous groups within thereference sequence. Preferably, residue positions which are notidentical differ by conservative amino acid substitutions. However,conservative substitutions are not included as a match when determiningsequence identity.

“Sequence homology”, as used herein, refers to a method of determiningthe relatedness of two sequences. To determine sequence homology, two ormore sequences are optimally aligned, and gaps are introduced ifnecessary. However, in contrast to “sequence identity”, conservativeamino acid substitutions are counted as a match when determiningsequence homology. In other words, to obtain a polypeptide orpolynucleotide having 95% sequence homology with a reference sequence,85%, preferably 90%, even more preferably 95% of the amino acid residuesor nucleotides in the reference sequence must match or comprise aconservative substitution with another amino acid or nucleotide, or anumber of amino acids or nucleotides up to 15%, preferably up to 10%,even more preferably up to 5% of the total amino acid residues ornucleotides, not including conservative substitutions, in the referencesequence may be inserted into the reference sequence. Preferably thehomologous sequence comprises at least a stretch of 50, even morepreferably 100, even more preferably 250, even more preferably 500nucleotides.

A “conservative substitution” refers to the substitution of an aminoacid residue or nucleotide with another amino acid residue or nucleotidehaving similar characteristics or properties including size,hydrophobicity, etc., such that the overall functionality does notchange significantly.

Isolated” means altered “by the hand of man” from its natural state,i.e., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, a polynucleotide orpolypeptide naturally present in a living organism is not “isolated,”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, as the term is employedherein.

Thus, a further aspect of the present invention relates to animmunogenic composition effective for lessening the severity of clinicalsymptoms associated with PCV2 infection comprising PCV2 ORF2 protein.Preferably, the PCV2 ORF2 protein is anyone of those, described above.Preferably, said PCV2 ORF2 protein is

-   -   i) a polypeptide comprising the sequence of SEQ ID NO: 5, SEQ ID        NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11;    -   ii) any polypeptide that is at least 80% homologous to the        polypeptide of i),    -   iii) any immunogenic portion of the polypeptides of i) and/or        ii)    -   iv) the immunogenic portion of iii), comprising at least 10        contiguous amino acids included in the sequences of SEQ ID NO:        5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11,    -   v) a polypeptide that is encoded by a DNA comprising the        sequence of SEQ ID NO: 3 or SEQ ID NO: 4.    -   vi) any polypeptide that is encoded by a polynucleotide that is        at least 80% homologous to the polynucleotide of v),    -   vii) any immunogenic portion of the polypeptides encoded by the        polynucleotide of v) and/or vi)    -   viii) the immunogenic portion of vii), wherein the        polynucleotide coding for said immunogenic portion comprises at        least 30 contiguous nucleotides included in the sequences of SEQ        ID NO: 3, or SEQ ID NO: 4.

Preferably any of those immunogenic portions will have the immunogeniccharacteristics of PCV2 ORF2 protein that is encoded by the sequence ofSEQ ID NO: 3 or SEQ ID NO: 4.

According to a further aspect, PCV2 ORF2 protein is provided in theimmunological composition at an antigen inclusion level effective forinducing the desired immune response, namely reducing the incidence ofor lessening the severity of clinical signs resulting from PCV2infection. Preferably, the PCV2 ORF2 protein inclusion level is at least0.2 μg antigen/ml of the final immunogenic composition (μg/ml), morepreferably from about 0.2 to about 400 μg/ml, still more preferably fromabout 0.3 to about 200 μg/ml, even more preferably from about 0.35 toabout 100 μg/ml, still more preferably from about 0.4 to about 50 μg/ml,still more preferably from about 0.45 to about 30 μg/ml, still morepreferably from about 0.6 to about 15 μg/ml, even more preferably fromabout 0.75 to about 8 μg/ml, even more preferably from about 1.0 toabout 6 μg/ml, still more preferably from about 1.3 to about 3.0 μg/ml,even more preferably from about 1.4 to about 2.5 μg/ml, even morepreferably from about 1.5 to about 2.0 μg/ml, and most preferably about1.6 μg/ml.

According to a further aspect, the ORF2 antigen inclusion level is atleast 0.2 μg PCV2 ORF2 protein, as described above, per dose of thefinal antigenic composition (μg/dose), more preferably from about 0.2 toabout 400 μg/dose, still more preferably from about 0.3 to about 200μg/dose, even more preferably from about 0.35 to about 100 μg/dose,still more preferably from about 0.4 to about 50 μg/dose, still morepreferably from about 0.45 to about 30 μg/dose, still more preferablyfrom about 0.6 to about 15 μg/dose, even more preferably from about 0.75to about 8 μg/dose, even more preferably from about 1.0 to about 6μg/dose, still more preferably from about 1.3 to about 3.0 μg/dose, evenmore preferably from about 1.4 to about 2.5 μg/dose, even morepreferably from about 1.5 to about 2.0 μg/dose, and most preferablyabout 1.6 μg/dose.

The PCV2 ORF2 polypeptide used in an immunogenic composition inaccordance with the present invention can be derived in any fashionincluding isolation and purification of PCV2 ORF2, standard proteinsynthesis, and recombinant methodology. Preferred methods for obtainingPCV2 ORF2 polypeptide are described herein above and are also providedin U.S. patent application Ser. No. 11/034,797, the teachings andcontent of which are hereby incorporated by reference. Briefly,susceptible cells are infected with a recombinant viral vectorcontaining PCV2 ORF2 DNA coding sequences, PCV2 ORF2 polypeptide isexpressed by the recombinant virus, and the expressed PCV2 ORF2polypeptide is recovered from the supernate by filtration andinactivated by any conventional method, preferably using binaryethylenimine, which is then neutralized to stop the inactivationprocess.

Thus, according to a further aspect the immunogenic compositioncomprises i) any of the PCV2 ORF2 protein described above, preferably inconcentrations described above, and ii) at least a portion of the viralvector expressing said PCV2 ORF2 protein, preferably of a recombinantbaculovirus. Moreover, according to a further aspect, the immunogeniccomposition comprises i) any of the PCV2 ORF2 protein described above,preferably in concentrations described above, ii) at least a portion ofthe viral vector expressing said PCV2 ORF2 protein, preferably of arecombinant baculovirus, and iii) a portion of the cell culturesupernate.

According to one specific embodiment of the production and recoveryprocess for PCV2 ORF2 protein, the cell culture supernate is filteredthrough a membrane having a pore size, preferably between about 0.45 to1 μm. Thus, a further aspect relates to an immunogenic composition thatcomprises i) any of the PCV2 ORF2 protein described above, preferably inconcentrations described above, ii) at least a portion of the viralvector expressing said PCV2 ORF2 protein, preferably of a recombinantbaculovirus, and iii) a portion of the cell culture; wherein about 90%of the components have a size smaller than 1 μm.

According to a further aspect, the present invention relates to animmunogenic composition that comprises i) any of the PCV2 ORF2 proteindescribed above, preferably in concentrations described above, ii) atleast a portion of the viral vector expressing said PCV2 ORF2 protein,iii) a portion of the cell culture, iv) and inactivating agent toinactivate the recombinant viral vector preferably BEI, wherein about90% of the components i) to iii) have a size smaller than 1 μm.Preferably, BEI is present in concentrations effective to inactivate thebaculovirus. Effective concentrations are described above.

According to a further aspect, the present invention relates to animmunogenic composition that comprises i) any of the PCV2 ORF2 proteindescribed above, preferably in concentrations described above, ii) atleast a portion of the viral vector expressing said PCV2 ORF2 protein,iii) a portion of the cell culture, iv) an inactivating agent toinactivate the recombinant viral vector preferably BEI, and v) anneutralization agent to stop the inactivation mediated by theinactivating agent, wherein about 90% of the components i) to iii) havea size smaller than 1 μm. Preferably, if the inactivating agent is BEI,said composition comprises sodium thiosulfate in equivalent amounts toBEI.

The polypeptide is incorporated into a composition that can beadministered to an animal susceptible to PCV2 infection. In preferredforms, the composition may also include additional components known tothose of skill in the art (see also Remington's Pharmaceutical Sciences.(1990). 18th ed. Mack Publ., Easton). Additionally, the composition mayinclude one or more veterinary-acceptable carriers. As used herein, “aveterinary-acceptable carrier” includes any and all solvents, dispersionmedia, coatings, adjuvants, stabilizing agents, diluents, preservatives,antibacterial and antifungal agents, isotonic agents, adsorptiondelaying agents, and the like.

In a preferred embodiment, the immunogenic composition comprises PCV2ORF2 protein as provided herewith, preferably in concentrationsdescribed above as an antigenic component, which is mixed with anadjuvant, preferably Carbopol, and physiological saline.

Those of skill in the art will understand that the composition hereinmay incorporate known injectable, physiologically acceptable, sterilesolutions. For preparing a ready-to-use solution for parenteralinjection or infusion, aqueous isotonic solutions, such as e.g. salineor corresponding plasma protein solutions are readily available. Inaddition, the immunogenic and vaccine compositions of the presentinvention can include diluents, isotonic agents, stabilizers, oradjuvants. Diluents can include water, saline, dextrose, ethanol,glycerol, and the like. Isotonic agents can include sodium chloride,dextrose, mannitol, sorbitol, and lactose, among others. Stabilizersinclude albumin and alkali salts of ethylendiamintetracetic acid, amongothers. Suitable adjuvants, are those described above. Most preferred isthe use of Carbopol, in particular the use of Carbopol 971P, preferablyin amounts as described above (e.g. of about 500 μg to about 5 mg perdose, even more preferably in an amount of about 750 μg to about 2.5 mgper dose and most preferably in an amount of about 1 mg per dose).

Thus, the present invention also relates to an immunogenic compositionthat comprises i) any of the PCV2 ORF2 proteins described above,preferably in concentrations described above, ii) at least a portion ofthe viral vector expressing said PCV2 ORF2 protein, iii) a portion ofthe cell culture, iv) an inactivating agent to inactivate therecombinant viral vector, preferably BEI, and v) a neutralization agentto stop the inactivation mediated by the inactivating agent, preferablysodium thiosulfate in equivalent amounts to BEI; and vi) a suitableadjuvant, preferably Carbopol 971, in amounts described above; whereinabout 90% of the components i) to iii) have a size smaller than 1 μm.According to a further aspect, this immunogenic composition furthercomprises a pharmaceutical acceptable salt, preferably a phosphate saltin physiologically acceptable concentrations. Preferably, the pH of saidimmunogenic composition is adjusted to a physiological pH, meaningbetween about 6.5 and 7.5.

Thus, the present invention also relates to an immunogenic compositioncomprises per one ml i) at least 1.6 μg of PCV2 ORF2 protein describedabove, ii) at least a portion of baculovirus expressing said PCV2 ORF2protein iii) a portion of the cell culture, iv) about 2 to 8 mM BEI, v)sodium thiosulfate in equivalent amounts to BEI; and vi) about 1 mgCarbopol 971, and vii) phosphate salt in a physiologically acceptableconcentration; wherein about 90% of the components i) to iii) have asize smaller than 1 μm and the pH of said immunogenic composition isadjusted to about 6.5 to 7.5.

The immunogenic compositions can further include one or more otherimmunomodulatory agents such as, e. g., interleukins, interferons, orother cytokines. The immunogenic compositions can also includeGentamicin and Merthiolate. While the amounts and concentrations ofadjuvants and additives useful in the context of the present inventioncan readily be determined by the skilled artisan, the present inventioncontemplates compositions comprising from about 50 μg to about 2000 μgof adjuvant and preferably about 250 μg/ml dose of the vaccinecomposition. In another preferred embodiment, the present inventioncontemplates vaccine compositions comprising from about 1 ug/ml to about60 μg/ml of antibiotics, and more preferably less than about 30 μg/ml ofantibiotics.

Thus, the present invention also relates to an immunogenic compositionthat comprises i) any of the PCV2 ORF2 proteins described above,preferably in concentrations described above, ii) at least a portion ofthe viral vector expressing said PCV2 ORF2 protein, iii) a portion ofthe cell culture, iv) an inactivating agent to inactivate therecombinant viral vector preferably BEI, and v) an neutralization agentto stop the inactivation mediated by the inactivating agent, preferablysodium thiosulfate in equivalent amounts to BEI; vi) a suitableadjuvant, preferably Carbopol 971 in amounts described above; vii) apharmaceutical acceptable concentration of a saline buffer, preferablyof a phosphate salt, and viii) an anti-microbiological active agent;wherein about 90% of the components i) to have a size smaller than 1 μm.

It has been surprisingly found, that the immunogenic compositionprovided herewith comprises was highly stable over a period of 24months. It has also been found the immunogenic compositions providedherewith, comprising recombinant, baculovirus expressed PCV2 ORF2protein as provided herewith are very effective in reducing the clinicalsymptoms associated with PCV2 infections. It has been surprisinglyfound, that the immunogenic compositions comprising the recombinantbaculovirus expressed PCV2 ORF2 protein as provided herewith, are moreeffective than an immunogenic composition comprising the whole PCV2virus in an inactivated form, or isolated viral PCV2 ORF2 antigen. Inparticular, it has been surprisingly found, that the recombinantbaculovirus expressed PCV2 ORF2 protein is effective is in very lowconcentrations, which means in concentrations up to 0.25 μg/dose. Thisunexpected high immunogenic potential of the PCV2 ORF2 protein could befurther increased by the addition of Carbopol.

A further aspect relates to a container comprising at least one dose ofthe immunogenic composition of PCV2 ORF2 protein as provided herewith,wherein one dose comprises at least 2 μg PCV2 ORF2 protein, preferably 2to 16 μg PCV2 ORF2 protein. Said container can comprise from 1 to 250doses of the immunogenic composition, preferably it contains 1, 10, 25,50, 100, 150, 200, or 250 doses of the immunogenic composition of PCV2ORF2 protein. Preferably, each of the containers comprising more thanone dose of the immunogenic composition of PCV2 ORF2 protein furthercomprises an anti-microbiological active agent. Those agents are forexample, antibiotics including Gentamicin and Merthiolate and the like.Thus, one aspect of the present invention relates to a container thatcomprises from 1 to 250 doses of the immunogenic composition of PCV2ORF2 protein, wherein one dose comprises at least 2 μg PCV2 ORF2protein, and Gentamicin and/or Merthiolate, preferably from about 1μg/ml to about 60 μg/ml of antibiotics, and more preferably less thanabout 30 μg/ml.

A further aspect relates to a kit, comprising any of the containers,described above, and an instruction manual, including the informationfor the intramuscular application of at least one dose of theimmunogenic composition of PCV2 ORF2 protein into piglets to lessen theseverity of clinical symptoms associated with PCV2 infection. Moreover,according to a further aspect, said instruction manual comprises theinformation of a second or further administration(s) of at least onedose of the immunogenic composition of PCV2 ORF2, wherein the secondadministration or any further administration is at least 14 days beyondthe initial or any former administration. Preferably, said instructionmanual also includes the information, to administer an immune stimulant.Preferably, said immune stimulant shall be given at least twice.Preferably, at least 3, more preferably at least 5, and even morepreferably at least 7 days are between the first and the second or anyfurther administration of the immune stimulant. Preferably, the immunestimulant is given at least 10 days, preferably 15, even more preferably20, and still even more preferably at least 22 days beyond the initialadministration of the immunogenic composition of PCV2 ORF2 protein. Apreferred immune stimulant is for example is keyhole limpet hemocyanin(KLH), preferably emulsified with incomplete Freund's adjuvant(KLH/ICFA). However, it is herewith understood, that any other immunestimulant known to a person skilled in the art can also be used. “Immunestimulant” as used herein, means any agent or composition that cantrigger a general immune response, preferably without initiating orincreasing a specific immune response, for example the immune responseagainst a specific pathogen. It is further instructed to administer theimmune stimulant in a suitable dose. Moreover, the kit may also comprisea container, including at least one dose of the immune stimulant,preferably one dose of KLH, or KLH/ICFA.

Moreover, it has also been surprisingly found that the immunogenicpotential of the immunogenic compositions comprising recombinantbaculovirus expressed PCV2 ORF2 protein, preferably in combination withCarbopol, can be further enhanced by the administration of the IngelVacPRRS MLV vaccine (see Example 5). PCV2 clinical signs and diseasemanifestations are greatly magnified when PRRS infection is present.However, the immunogenic compositions and vaccination strategies asprovided herewith lessened this effect greatly, and more than expected.In other words, an unexpected synergistic effect was observed whenanimals, preferably pigs, are treated with any of the PCV2 ORF2immunogenic compositions, as provided herewith, and the Ingelvac PRRSMLV vaccine (Boehringer Ingelheim).

Thus, a further aspect of the present invention relates to the kit asdescribed above, comprising the immunogenic composition of PCV2 ORF2 asprovided herewith and the instruction manual, wherein the instructionmanual further includes the information to administer the PCV2 ORF2immunogenic composition together, or around the same time as, with animmunogenic composition that comprises PRRS antigen, preferablyadjuvanted PRRS antigen. Preferably, the PRRS antigen is IngelVac® PRRSMLV (Boehringer Ingelheim).

A further aspect of the present invention also relates to a kitcomprising i) a container containing at least one dose of an immunogeniccomposition of PCV2 ORF2 as provided herewith, and ii) a containercontaining an immunogenic composition comprising PRRS antigen,preferably adjuvanted PRRS antigen. Preferably the PRRS antigen isIngelVac® PRRS MLV (Boehringer Ingelheim). More preferably, the kitfurther comprises an instruction manual, including the information toadminister both pharmaceutical compositions. Preferably, it contains theinformation that the PCV2 ORF2 containing composition is administeredtemporally prior to the PRRS containing composition.

A further aspect, relates to the use of any of the compositions providedherewith as a medicament, preferably as a veterinary medicament, evenmore preferably as a vaccine. Moreover, the present invention alsorelates to the use of any of the compositions described herein, for thepreparation of a medicament for lessening the severity of clinicalsymptoms associated with PCV2 infection. Preferably, the medicament isfor the prevention of a PCV2 infection, even more preferably in piglets.

A further aspect relates to a method for (i) the prevention of aninfection, or re-infection with PCV2 or (ii) the reduction orelimination of clinical symptoms caused by PCV2 in a subject, comprisingadministering any of the immunogenic compositions provided herewith to asubject in need thereof. Preferably, the subject is a pig. Preferably,the immunogenic composition is administered intramuscularly. Preferably,one dose or two doses of the immunogenic composition is/areadministered, wherein one dose preferably comprises at least about 2 μgPCV2 ORF2 protein, even more preferably about 2 to about 16 μg, and atleast about 0.1 to about 5 mg Carbopol, preferably about 1 mg Carbopol.A further aspect relates to the method of treatment as described above,wherein a second application of the immunogenic composition isadministered. Preferably, the second administration is done with thesame immunogenic composition, preferably having the same amount of PCV2ORF2 protein. Preferably the second administration is also givenintramuscularly. Preferably, the second administration is done at least14 days beyond the initial administration, even more preferably at least4 weeks beyond the initial administration.

According to a further aspect, the method of treatment also comprisesthe administration of an immune stimulant. Preferably, said immunestimulant is administered at least twice. Preferably, at least 3, morepreferably at least 5 days, even more preferably at least 7 days arebetween the first and the second administration of the immune stimulant.Preferably, the immune stimulant is administered at least 10 days,preferably 15, even more preferably 20, still more preferably at least22 days beyond the initial administration of the PCV2 ORF2 immunogeniccomposition. A preferred immune stimulant is for example is keyholelimpet hemocyanin (KLH), still preferably emulsified with incompleteFreund's adjuvant (KLH/ICFA). However, it is herewith understood, thatany other immune stimulant known to a person skilled in the art can alsobe used. It is within the general knowledge of a person skilled in theart to administer the immune stimulant in a suitable dose.

According to a further aspect, the method of treatments described abovealso comprises the administration of PRRS antigen. Preferably the PRRSantigen is IngelVac® PRRS MLV (Boehringer Ingelheim). Preferably, saidPRRS antigen is administered temporally beyond the administration of theimmunogenic composition of PCV2 ORF2 protein.

According to a further aspect, the present invention provides amultivalent combination vaccine which includes an immunological agenteffective for reducing the incidence of or lessening the severity ofPCV2 infection, and at least one immunogenic active component againstanother disease-causing organism in swine.

In particular the immunological agent effective for reducing theincidence of or lessening the severity of PCV2 infection is a PCV2antigen. Preferably, said PCV2 antigen is a PCV2 ORF2 protein asprovided herewith, or any immunogenic composition as described above,that comprises PCV2 ORF2 protein.

However it is herewith understood, that a PCV2 antigen also refers toany composition of matter that comprises at least one antigen that caninduce, stimulate or enhance the immune response against PCV2 infection,when administered to a pig. Preferably, said PCV2 antigen is the wholePCV2 virus, preferably in an inactivated form, a life modified orattenuated PCV2 virus, a chimeric virus that comprises at least animmunogenic amino acid sequence of PCV2, any other polypeptide orcomponent that comprises at least an immunogenic amino acid sequence ofPCV2. The terms “immunogenic protein”, “immunogenic polypeptide” or“immunogenic amino acid sequence” as used herein refer to any amino acidsequence which elicits an immune response in a host against a pathogencomprising said immunogenic protein, immunogenic polypeptide orimmunogenic amino acid sequence. An “immunogenic protein”, “immunogenicpolypeptide” or “immunogenic amino acid sequence” as used herein,includes the full-length sequence of any proteins, analogs thereof, orimmunogenic fragments thereof. By “immunogenic fragment” is meant afragment of a protein which includes one or more epitopes and thuselicits the immunological response against the relevant pathogen. Suchfragments can be identified using any number of epitope mappingtechniques, well known in the art. See, e.g., Epitope Mapping Protocolsin Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996)Humana Press, Totowa, N.J. For example, linear epitopes may bedetermined by e.g., concurrently synthesizing large numbers of peptideson solid supports, the peptides corresponding to portions of the proteinmolecule, and reacting the peptides with antibodies while the peptidesare still attached to the supports. Such techniques are known in the artand described in, e.g., U.S. Pat. No. 4,708,871; Geysen et al. (1984)Proc. Natl. Acad. Sci. USA 81:3998-4002; Geysen et al. (1986) Molec.Immunol. 23:709-715. Similarly, conformational epitopes are readilyidentified by determining spatial conformation of amino acids such asby, e.g., x-ray crystallography and 2-dimensional nuclear magneticresonance See, e.g., Epitope Mapping Protocols, supra. Syntheticantigens are also included within the definition, for example,polyepitopes, flanking epitopes, and other recombinant or syntheticallyderived antigens. See, e.g., Bergmann et al. (1993) Eur. J. Immunol.23:2777-2781; Bergmann et al. (1996), J. Immunol. 157:3242-3249;Suhrbier, A. (1997), Immunol. and Cell Biol. 75:402-408; Gardner et al.,(1998) 12th World AIDS Conference, Geneva, Switzerland, June 28-Jul. 3,1998.

According to further embodiment, said PCV-2 antigen is Inglevac®CircoFLEX™ (Boehringer Ingelheim Vetmedica Inc, St Joseph, Mo., USA),CircoVac® (Merial SAS, Lyon, France), CircoVent (Intervet Inc.,Millsboro, Del., USA), or Suvaxyn PCV-2 One Dose® (Fort Dodge AnimalHealth, Kansas City, Kans., USA).

An “immunological or immune response” to a composition or vaccine is thedevelopment in the host of a cellular and/or antibody-mediated immuneresponse to the composition or vaccine of interest. Usually, an “immuneresponse” includes but is not limited to one or more of the followingeffects: the production or activation of antibodies, B cells, helper Tcells, suppressor T cells, and/or cytotoxic T cells and/or yd T cells,directed specifically to an antigen or antigens included in thecomposition or vaccine of interest. Preferably, the host will displayeither a therapeutic or protective immunological response such thatresistance to new infection will be enhanced and/or the clinicalseverity of the disease reduced. Such protection will be demonstrated byeither a reduction or lack of the symptoms associated with hostinfections as described above.

Preferably the other disease-causing organism in swine is selected fromthe group consisting of: Actinobacillus pleuropneumonia (1); Adenovirus(2); Alphavirus such as Eastern equine encephalomyelitis viruses (3);Bordetella bronchiseptica (4); Brachyspira spp. (5), preferably B.hyodyentheriae (6); B. piosicoli (7), Brucella suis, preferably biovars1, 2, and 3 (8); Clasical swine fever virus (9); Clostridium spp. (10),preferably Cl. difficile (11), Cl. perfringens types A, B, and C (12),Cl. novyi (13), Cl. septicum (14), Cl. tetani (15); Coronavirus (16),preferably Porcine Respiratory Corona virus (17); Eperythrozoonosis suis(18); Erysipelothrix rhsiopathiae (19) Escherichia coli (20);Haemophilus parasuis, preferably subtypes 1, 7 and 14 (21)Hemagglutinating encephalomyelitis virus (22); Japanese EncephalitisVirus (23); Lawsonia intracellularis(24) Leptospira spp. (25),preferably Leptospira australis (26); Leptospira canicola (27);Leptospira grippotyphosa (28); Leptospira icterohaemorrhagicae (29); andLeptospira interrogans (30); Leptospira pomona (31); Leptospiratarassovi (32); Mycobacterium spp. (33) preferably M. avium (34), M.intracellulare (35) and M. bovis (36); Mycoplasma hyopneumoniae (M. hyo)(37) Pasteurella multocida (38); Porcine cytomegalovirus (39); PorcineParvovirus (40); Porcine Reproductive and Respiratory Syndrome (PRRS)Virus (41) Pseudorabies virus (42); Rotavirus (43); Salmonella spp.(44), preferably S. thyhimurium (45) and S. choleraesuis (46); Staph.hyicus (47); Staphylococcus spp. (48) preferably Streptococcus spp.(49), preferably Strep. suis (50); Swine herpes virus (51); SwineInfluenza Virus (52); Swine pox virus (53); Swine pox virus (54);Vesicular stomatitis virus (55); Virus of vesicular exanthema of swine(56); Leptospira Hardjo (57); and/or Mycoplasma hyosynoviae (58).

Any reference made in connection with a swine pathogen in the followingcan be made by naming the pathpgen, for example M. hyo, or by makingreference to the number in 0 behind the pathogen, that is found above.For example reference to M. hyo can be made by M. hyo or by (37).

Thus, the present invention relates to a combination vaccine for thetreatment and/or prophylaxis of swine, that includes an immunologicalagent effective for reducing the incidence of or lessening the severityof PCV2 infection, preferably a PCV2 antigen, and further animmunological active component effective for the treatment and/orprophylaxis of infections caused by any of the swine pathogens (1), (2),(3), (4), (5), (6), (7), (8), (9), (10), (11), (12), (13), (14), (15),(16), (17), (18), (19), (20), (21), (22), (23), (24), (25), (26), (27),(28), (29), (30), (31), (32), (33), (34), (35), (36), (37), (38), (39),(40), (41), (42), (43), (44), (45), (46), (47), (48), (49), (50), (51),(52), (53), (54), (55), (56), (57) and/or (58), or is an immunologicalactive component of said swine pathogen(s). [combo 1].

An “immunological active component” as used herein means a componentthat induces or stimulates the immune response in an animal to whichsaid component is administered. According to a preferred embodiment,said immune response is directed to said component or to a microorganismcomprising said component. According to a further preferred embodiment,the immunological active component is an attenuated microorganism,including modified live virus (MLV), a killed-microorganism or at leastan immunological active part of a microorganism.

“Immunological active part of a microorganism” as used herein means aprotein-, sugar-, and or glycoprotein containing fraction of amicroorganism that comprises at least one antigen that induces orstimulates the immune response in an animal to which said component isadministered. According to a preferred embodiment, said immune responseis directed to said immunological active part of a microorganism or to amicroorganism comprising said immunological active part.

Preferably the further immunological active component of [combo 1] iseffective for the treatment and/or prophylaxis of infections caused bythe swine pathogen (41) or is an immunological active component of theswine pathogen (41).

According to another aspect, the further immunological active componentof [combo 1] is effective for the treatment and/or prophylaxis ofinfections caused by the swine pathogen (37) or is an immunologicalactive component of the swine pathogen (37).

According to another aspect, the further immunological active componentof [combo 1] is effective for the treatment and/or prophylaxis ofinfections caused by the swine pathogen (1) or is an immunologicalactive component of the swine pathogen (1).

According to another aspect, the further immunological active componentof [combo 1] is effective for the treatment and/or prophylaxis ofinfections caused by the swine pathogen (7) or is an immunologicalactive component of the swine pathogen (7).

According to another aspect, the further immunological active componentof [combo 1] is effective for the treatment and/or prophylaxis ofinfections caused by the swine pathogen (24) or is an immunologicalactive component of the swine pathogen (24).

According to another aspect, the further immunological active componentof [combo 1] is effective for the treatment and/or prophylaxis ofinfections caused by the swine pathogen (38) or is an immunologicalactive component of the swine pathogen (38).

According to another aspect, the further immunological active componentof [combo 1] is effective for the treatment and/or prophylaxis ofinfections caused by the swine pathogen (21) or is an immunologicalactive component of the swine pathogen (21).

According to another aspect, the further immunological active componentof [combo 1] is effective for the treatment and/or prophylaxis ofinfections caused by the swine pathogen (40) or is an immunologicalactive component of the swine pathogen (40).

According to another aspect, the further immunological active componentof [combo 1] is effective for the treatment and/or prophylaxis ofinfections caused by the swine pathogen (2) or is an immunologicalactive component of the swine pathogen (2).

According to another aspect, the further immunological active componentof [combo 1] is effective for the treatment and/or prophylaxis ofinfections caused by the swine pathogen (44) or is an immunologicalactive component of the swine pathogen (44).

According to another aspect, the further immunological active componentof [combo 1] is effective for the treatment and/or prophylaxis ofinfections caused by the swine pathogen (50) or is an immunologicalactive component of the swine pathogen (50).

According to another aspect, the further immunological active componentof [combo 1] is effective for the treatment and/or prophylaxis ofinfections caused by the swine pathogen (19), preferably (20) and/or(21) or is an immunological active component of the swine pathogen (19),preferably (20) and/or (21).

According to another aspect, the further immunological active componentof [combo 1] is effective for the treatment and/or prophylaxis ofinfections caused by the swine pathogen (22) or is an immunologicalactive component of the swine pathogen (22).

According to another aspect, the further immunological active componentof [combo 1] is effective for the treatment and/or prophylaxis ofinfections caused by the swine pathogens (41) and (37), or is animmunological active component of the swine pathogens (41) and (37).

According to another aspect, the further immunological active componentof [combo 1] is effective for the treatment and/or prophylaxis ofinfections caused by the swine pathogens (1) and (41), or is animmunological active component of the swine pathogens (1) and (41).

According to another aspect, the further immunological active componentof [combo 1] is effective for the treatment and/or prophylaxis ofinfections caused by the swine pathogens (1) and (37), or is animmunological active component of the swine pathogens (1) and (37).

According to another aspect, the further immunological active componentof [combo 1] is effective for the treatment and/or prophylaxis ofinfections caused by the swine pathogens, (1) (41) and (37), or is animmunological active component of the swine pathogens), (1), (41) and(37). In a preferred form, this combination is adjuvanted with Carbopol.

According to another aspect, the further immunological active componentof [combo 1] is effective for the treatment and/or prophylaxis ofinfections caused by the swine pathogens (1) and (21), or is animmunological active component of the swine pathogens (1) and (21).

According to another aspect, the further immunological active componentof [combo 1] is effective for the treatment and/or prophylaxis ofinfections caused by the swine pathogens (21) and (41), or is animmunological active component of the swine pathogens (21) and (41).

According to another aspect, the further immunological active componentof [combo 1] is effective for the treatment and/or prophylaxis ofinfections caused by the swine pathogens (21) and (37), or is animmunological active component of the swine pathogens (21) and (37).

According to another aspect, the further immunological active componentof [combo 1] is effective for the treatment and/or prophylaxis ofinfections caused by the swine pathogens (21) and (38), or is animmunological active component of the swine pathogens (21) and (38).

According to another aspect, the further immunological active componentof [combo 1] is effective for the treatment and/or prophylaxis ofinfections caused by the swine pathogens (7) and (19), or is animmunological active component of the swine pathogens (7) and (19).

According to another aspect, the further immunological active componentof [combo 1] is effective for the treatment and/or prophylaxis ofinfections caused by the swine pathogens (38) and (33), preferably (34),(35) and/or (36), or is an immunological active component of the swinepathogens (38) and (33) preferably (34), (35) and/or (36).

According to another aspect, the further immunological active componentof [combo 1] is effective for the treatment and/or prophylaxis ofinfections caused by the swine pathogens (49), preferably (50), and(21), or is an immunological active component of the swine pathogens(49) preferably (50), and (21).

According to another aspect, the further immunological active componentof [combo 1] is effective for the treatment and/or prophylaxis ofinfections caused by the swine pathogens (49) preferably (50), (20) and(21), or is an immunological active component of the swine pathogens(49) preferably (50), (20) and (21).

According to another aspect, the further immunological active componentof [combo 1] is effective for the treatment and/or prophylaxis ofinfections caused by the swine pathogens (49) preferably (50), (20) and(21), or is an immunological active component of the swine pathogens(49) preferably (50), (20) and (21).

According to another aspect, the further immunological active componentof [combo 1] is effective for the treatment and/or prophylaxis ofinfections caused by the swine pathogens (49), preferably (50), (20),(38) and (21), or is an immunological active component of the swinepathogens (49), preferably (50), (20), (38) and (21).

According to another aspect, the further immunological active componentof [combo 1] is effective for the treatment and/or prophylaxis ofinfections caused by the swine pathogens (49) preferably (50), (20),(33) and (21), or is an immunological active component of the swinepathogens (49) preferably (50), (20) (33) and (21).

According to another aspect, the further immunological active componentof [combo 1] is effective for the treatment and/or prophylaxis ofinfections caused by the swine pathogens (49), preferably (50), (20),(38) (33) and (21), or is an immunological active component of the swinepathogens (49), preferably (50), (20), (38), (33) and (21).

According to another aspect, the further immunological active componentof [combo 1] is effective for the treatment and/or prophylaxis ofinfections caused by the swine pathogens (41), (40), and (19), or is animmunological active component of the swine pathogens (41), (40), and(19).

According to another aspect, the further immunological active componentof [combo 1] is effective for the treatment and/or prophylaxis ofinfections caused by the swine pathogens (38), (4), and (19), or is animmunological active component of the swine pathogens (38), (4), and(19).

According to another aspect, the further immunological active componentof [combo 1] is effective for the treatment and/or prophylaxis ofinfections caused by the swine pathogens (38), (4), (21), and (19), oris an immunological active component of the swine pathogens (38), (4),(21) and (19).

According to another aspect, the further immunological active componentof [combo 1] is effective for the treatment and/or prophylaxis ofinfections caused by the swine pathogens (20), preferably, (20), (31)and (38), or is an immunological active component of the swine pathogens(20), (31), (38).

According to another aspect, the further immunological active componentof [combo 1] is effective for the treatment and/or prophylaxis ofinfections caused by the swine pathogens (5), preferably, (5) and (24),or is an immunological active component of the swine pathogens (5), and(24).

According to another aspect, the further immunological active componentof [combo 1] is effective for the treatment and/or prophylaxis ofinfections caused by the swine pathogens (1), preferably, (1), and (5),or is an immunological active component of the swine pathogens (1), and(5).

According to another aspect, the further immunological active componentof [combo 1] is effective for the treatment and/or prophylaxis ofinfections caused by the swine pathogens (41), preferably, (40), (27),(28), (29), (31), (19) and (59), or is an immunological active componentof the swine pathogens (41), (40), (27), (28), (29), (31), (19) and(57).

According to another aspect, the further immunological active componentof [combo 1] is effective for the treatment and/or prophylaxis ofinfections caused by the swine pathogens (6), preferably, (6), (19),(38) and (58) or is an immunological active component of the swinepathogens (1), and (5).

According to a further aspect, the further immunological activecomponent of the combination vaccine is selected from the groupconsisting Enterisol® Ileitis, Enterisol® Ileitis FF, Enterisol® SC-54,Enterisol® SC-54 FF, Enterisol® ERY-ALC, Ingelvac® APP ALC, Ingelvac®AR4, Ingelvac® HP-1, Ingelvac® HPE-1, Ingelvac® M. hyo, Ingelvac® PRRSMLV, Ingelvac® PRRS ATP, Ingelvac® PRV-G1, Reprocyc® PRRS PLE, Reprocyc®PLE, Tetguard™, Toxivac® AD+E, Toxivac® Plus Parsius, (all of BoehringerIngelheim, St. Joseph, Mo., USA); Circovent, Porcilis Coli, PorcilisERY+PARVO, Porcilis Ery, Porcilis Glasser, Porcilis Parvo, PorcilisPorcoli DF, Porcilis APP, Porcilis AR-T, Porcilis AR-T DF, PorcilisPorcoli, Porcilis Porcoli Diluvac forte, Porcilis PRRS, Porcilis Porcol5, Porcilis Aujeszky, Porcilis Begonia Diluvac, Porcilis BegoniaI.D.A.L., Porcilis Begonia Unisole, Porcilis M. hyo, Porcilis Atrinord,Myco Silencer® BPM, Myco Silencer® BPME, Myco Silencer® ME, MycoSilencer® M, Myco Silencer® Once, Myco Silencer® MEH, Rhinogen® BPE,Rhinogen® CTE 5000, Rhinogen® CTSE, Score, Sow Bac® E II, Sow Bac® CEII, Sow Bac® TREC, ProSystem® CE, ProSystem® RCE, ProSystem® TREC,ProSystem® Pillmune, ProSystem® Rotamune® with Imugan® II, ProSystem®Rota, ProSystem® Rotamune KV, ProSystem® TG-Emune® Rota with Imugan® II,ProSystem® TGE/Rota, ProSystem® TG-Emune® with Imugen®, ProSystem® TGE,MaGESTIC 7, MaGESTIC 8, MaGESTic™ with Spur®, MaGESTic® 7 with Spur®,MaGESTic® 8 with Spur®, End-FLUence® with Imugen® Il, End-FLUence® 2,PRRomiSE®, PRV-Begonia with Dlluvac Forte®, Argus® SC/ST, Strep Bac,Strep Bac® with Imugen® II, Colisorb, Heptavac, Lambivac, Porcovac plus,Erysorb Parvo all of Intervet Inc., Millsboro, Del., USA); Hyoresp,Circovac, Neocolipor, Parvoruvac, Parvosuin, Progressis, Viraflu, Akipor6.3, Jespur gl-, Jesflu gl- (all of Merial LTD, Duluth, Ga.); ER BAC®PLUS, ER BAC®, ER BAC® PLUS/LEPTOFERM-5®. ER BAC® Leptoferm-5®,Farrowsure®, Farrowsure® B, FARROWSURE® PLUS B, FARROWSURE® PLUS,FARROWSURE® PRV, FARROWSURE B-PRV, FLUSURE™, FLUSURE™ RTU, FLUSURE™/ERBAC® PLUS, FLUSURE™/ER BAC PLus®, FLUSURE™/RESPISURE®,FLUSURE™/RESPISURE® RTU, FLUSURE™/RESPISURE-ONC/ER BAC® PLUS,FLUSURE□/RespiSure 1 ONE®/ER BAC Plus®, FLUSURE™/RESPISURE ONE®,FLUSURE□/RESPISURE 1 ONE®, FLUSURE/Farrowsure Plus, FLUSURE/FarrowsurePlus B, LITTERGUARD® LT-C, LITTERGUARD® LT, PleuroGuard® 4, PneumosuisIII, Stellamune One, Stellamune Uno, Stellamune Once, Stellamune Mono,Stellamune Mycoplasma, Respisure One, Respisure®, Respisure 1 ONE®,Respisure 1 One®/ER Bac Plus®, Enduracell T, Zylexis (formerly known asBaypamune), Atrobac® 3, BratiVac®, BratiVac®-B,Leptoferm-5°°□□Parvo-Vac®/Leptoferm-5®, PR-Vac®-Killed, PR-Vac®, PR-VacPlus™□□ (all of Pfizer Inc., New York, N.Y., USA); Suvaxyn MH One,Suvaxyn RespiFend® MH, Suvaxyn Mycoplasma, Suvaxyn AujeszkyBartha+Diluent, Suvaxyn Aujeszky Bartha+o/w, Suvaxyn Aujeszky-Flu,Suvaxyn Aujeszky 783+o/w, Suvaxyn Ery, Suvaxyn Flu, Suvaxyn M. hyo,Suvaxyn MH-One, Suvaxyn Parvo ST, Suvaxyn Parvo/E, Suvaxyn RespiFend®APP, Suvaxyn RespiFend® HPS, Suvaxyn RespiFend® MH/HPS, SuvaxynRespiFend® MH, Suvaxyn® AR/T/E, Suvaxyn® EC-4, Suvaxyn® E, Suvaxyn®-E,Suvaxyn® E-oral, Suvaxyn® PLE, Suvaxyn® PLE/PrV gpl-, Suvaxyn® LE+B,Suvaxyn® PLE+B, Suvaxyn® PLE+B/PrV gpl-, Suvaxyn® SIV, Suvaxyn®SIV/Mh-one, Suvaxyn® P, Suvaxyn® PrV gpl-, Suvaxyn® PCV-2 One Shot (allof Fort Dodge Animal Health, Overland Park, Kans., USA (Wyeth);SCOURMUNE®, SCOURMUNE®-C, SCOURMUNE®-CR, AR-PAC®-PD+ER, AR-PARAPAC®+ER,M+Rhusigen®, M+PAC®, MaxiVac Excell®3, MaxiVac® H1N1, MaxiVac® H3N2,MaxiVac®-FLU, MaxiVac®-M+, MaxiVac Excel®, MaxiVac Excel 3, PARAPAC®,PNEU PAC®, PNEU PAC®-ER, PNEU PAC®+ER, PRV/Marker Gold®, PRV/MarkerGold®, PRV/Marker Gold®-MaxiVac® FLU, Rhusigen™, Gletvax 6, Covexin 8,M+PAC, Gletvax plus, M-Parapac™□□SS PAC® (all of Schering-Plough AnimalHealth Corporation, Kenilworth, N.J., USA); AMERVAC-PRRS, AUSKIPRA-BK,AUSKIPRA-GN, COLISUIN-CL, COLISUIN-TP, ERYSIPRAVAC, GRIPORK,HIPRASUIS-GLASSER, MYPRAVAC SUIS, NEUMOSUIN, PARVOSUIN, PARVOSUIN-MR,PARVOSUIN-MR/AD, RINIPRAVAC-DT, SUIPRAVAC-PRRS, SUIPRAVAC-RC, TOXIPRAPLUS (all of Laboratorios Hipra S.A., Amer, Girona, Spain); Clostricol,Coliporc Plus, Haeppovac, Per-C-Porc, Porciparvac, RESPIPORC ART+EP,RESPIPORC FLU, Respiporc M. HYO 1 SHOT, Rhusiovac,Rotlauf-Lebendimpfstoff, Salmoporc, Suisaloral, AK-vac MK35 (all of IDTImpfstoffwerk DessaTornau, Tornau, Germany); Mypravac Suis, (AlbrechtGmbH, Germany); Haemo Shield® P, Parapleuro Shield® P, ParapleuroShield® P+BE, Rhinicell® FD, Rhini Shield™ TX4, Prefarrow Shield® 9,Prefarrow Strep Shield®, Clostratox® BCD, Clostratox® C, Clostratox®Ultra C 1300, Porcine Ecolizer® 3+C, Porcine Pili Shield™□+C, PorcinePhi Shield™ . . . Porcine Ecolizer® 3, Ery Serum™□□Ery Shield™□□Ery VacOral, Ery Shield™+L5, PanSTAR™ Ery, Erycell™□□Parvo Shield® E, ParvoShield® L5E, Parvo Shield® L5, Parvo Shield®, Para Shield®, PneumoSTARSIV, PneumoSTAR™ Myco, Lepto Shield™ 5, Myco Shield™□□Salmo Shield® 2,Salmo Shield® Live, Amitox Tet™□□C. Perfingens Type A Toxoid (all ofNovartis Animal Health, Basel, Switzerland); Nitro-Sal (Akro); or anyantigen which in included in the compositions described above.Alternatively, when PCV2 antigen is already present in any of thosevaccines, (i) PCV2 antigen, as described herein, is added to any ofthose compostions/antigens, or (ii) the PCV2 antigen present in any ofthose vaccines is replaced by the PCV2 antigen, as described herein.

According to further aspect, the further immunological active componentof the combination vaccine is selected from the group consistingEnterisol® Ileitis, Enterisol® Ileitis FF, Enterisol® SC-54, Enterisol®SC-54 FF, Enterisol® ERY-ALC, Ingelvac® APP ALC, Ingelvac® AR4,Ingelvac® HP-1, Ingelvac® HPE-1, Ingelvac® M. hyo, Ingelvac® PRRS MLV,Ingelvac® PRRS ATP, Ingelvac® PRV-G1, Reprocyc® PRRS PLE, Reprocyc® PLE,Tetguard™, Toxivac® AD+E, Toxivac® Plus Parsius, (all of BoehringerIngelheim, St. Joseph, Mo., USA); Circovent, Porcilis Coli, PorcilisERY+PARVO, Porcilis Ery, Porcilis Glasser, Porcilis Parvo, PorcilisPorcoli DF, Porcilis APP, Porcilis AR-T, Porcilis AR-T DF, PorcilisPorcoli, Porcilis Porcoli Diluvac forte, Porcilis PRRS, Porcilis Porcol5, Porcilis Aujeszky, Porcilis Begonia Diluvac, Porcilis BegoniaI.D.A.L., Porcilis Begonia Unisole, Porcilis M. hyo, Porcilis Atrinord,Myco Silencer® BPM, Myco Silencer® BPME, Myco Silencer® ME, MycoSilencer® M, Myco Silencer® Once, Myco Silencer® MEH, Rhinogen® BPE,Rhinogen® CTE 5000, Rhinogen® CTSE, Score, Sow Bac® E II, Sow Bac® CEII, Sow Bac® TREC, ProSystem® CE, ProSystem® RCE, ProSystem® TREC,ProSystem® Pillmune, ProSystem® Rotamune® with Imugan® II, ProSystem®Rota, ProSystem® Rotamune KV, ProSystem® TG-Emune® Rota with Imugan® II,ProSystem® TGE/Rota, ProSystem® TG-Emune® with Imugen®, ProSystem® TGE,MaGESTIC 7, MaGESTIC 8, MaGESTic™ with Spur®, MaGESTic® 7 with Spur®,MaGESTic® 8 with Spur®, End-FLUence® with Imugen® Il, End-FLUence® 2,PRRomiSE®, PRV-Begonia with Dlluvac Forte®, Argus® SC/ST, Strep Bac,Strep Bac® with Imugen® II, Colisorb, Heptavac, Lambivac, Porcovac plus,Erysorb Parvo all of Intervet Inc., Millsboro, Del., USA); Hyoresp,Circovac, Neocolipor, Parvoruvac, Parvosuin, Progressis, Viraflu, Akipor6.3, Jespur gl-, Jesflu gl- (all of Merial LTD, Duluth, Ga.); ER BAC®PLUS, ER BAC®, ER BAC® PLUS/LEPTOFERM-5®′ ER BAC® Leptoferm-5®,Farrowsure®, Farrowsure® B, FARROWSURE® PLUS B, FARROWSURE® PLUS,FARROWSURE® PRV, FARROWSURE B-PRV, FLUSURE™, FLUSURE™ RTU, FLUSURE™/ERBAC® PLUS, FLUSURE™/ER BAC PLus®, FLUSURE™/RESPISURE®,FLUSURE™/RESPISURE® RTU, FLUSURE™/RESPISURE-ONC/ER BAC® PLUS,FLUSURE□/RespiSure 1 ONE®/ER BAC Plus®, FLUSURE™/RESPISURE ONE®,FLUSURE□/RESPISURE 1 ONE®, FLUSURE/Farrowsure Plus, FLUSURE/FarrowsurePlus B, LITTERGUARD® LT-C, LITTERGUARD® LT, PleuroGuard® 4, PneumosuisIII, Stellamune One, Stellamune Uno, Stellamune Once, Stellamune Mono,Stellamune Mycoplasma, Respisure One, Respisure®, Respisure 1 ONE®,Respisure 1 One®/ER Bac Plus®, Enduracell T, Zylexis (formerly known asBaypamune), Atrobac® 3, BratiVac®, BratiVac®-B,Leptoferm-5°°□□Parvo-Vac®/Leptoferm-5®, PR-Vac®-Killed, PR-Vac®, PR-VacPlus™□□(all of Pfizer Inc., New York, N.Y., USA); Suvaxyn MH One,Suvaxyn RespiFend® MH, Suvaxyn Mycoplasma, Suvaxyn AujeszkyBartha+Diluent, Suvaxyn Aujeszky Bartha+o/w, Suvaxyn Aujeszky-Flu,Suvaxyn Aujeszky 783+o/w, Suvaxyn Ery, Suvaxyn Flu, Suvaxyn M. hyo,Suvaxyn MH-One, Suvaxyn Parvo ST, Suvaxyn Parvo/E, Suvaxyn RespiFend®APP, Suvaxyn RespiFend® HPS, Suvaxyn RespiFend® MH/HPS, SuvaxynRespiFend® MH, Suvaxyn® AR/T/E, Suvaxyn® EC-4, Suvaxyn® E, Suvaxyn®-E,Suvaxyn® E-oral, Suvaxyn® PLE, Suvaxyn® PLE/PrV gpl-, Suvaxyn® LE+B,Suvaxyn® PLE+B, Suvaxyn® PLE+B/PrV gpl-, Suvaxyn® SIV, Suvaxyn®SIV/Mh-one, Suvaxyn® P, Suvaxyn® PrV gpl-, Suvaxyn® PCV-2 One Shot (allof Fort Dodge Animal Health, Overland Park, Kans., USA (Wyeth);SCOURMUNE®, SCOURMUNE®-C, SCOURMUNE®-CR, AR-PAC®-PD+ER, AR-PARAPAC®+ER,M+Rhusigen®, M+PAC®, MaxiVac Excell®3, MaxiVac® H1N1, MaxiVac® H3N2,MaxiVac®-FLU, MaxiVac®-M+, MaxiVac Excel®, MaxiVac Excel 3, PARAPAC®,PNEU PAC®, PNEU PAC®-ER, PNEU PAC®+ER, PRV/Marker Gold®, PRV/MarkerGold®, PRV/Marker Gold®-MaxiVac® FLU, Rhusigen™, Gletvax 6, Covexin 8,M+PAC, Gletvax plus, M-Parapac™□□SS PAC® (all of Schering-Plough AnimalHealth Corporation, Kenilworth, N.J., USA); AMERVAC-PRRS, AUSKIPRA-BK,AUSKIPRA-GN, COLISUIN-CL, COLISUIN-TP, ERYSIPRAVAC, GRIPORK,HIPRASUIS-GLASSER, MYPRAVAC SUIS, NEUMOSUIN, PARVOSUIN, PARVOSUIN-MR,PARVOSUIN-MR/AD, RINIPRAVAC-DT, SUIPRAVAC-PRRS, SUIPRAVAC-RC, TOXIPRAPLUS (all of Laboratorios Hipra S.A., Amer, Girona, Spain); Clostricol,Coliporc Plus, Haeppovac, Per-C-Porc, Porciparvac, RESPIPORC ART+EP,RESPIPORC FLU, Respiporc M. HYO 1 SHOT, Rhusiovac,Rotlauf-Lebendimpfstoff, Salmoporc, Suisaloral, AK-vac MK35 (all of IDTImpfstoffwerk DessaTornau, Tornau, Germany); Mypravac Suis, (AlbrechtGmbH, Germany); Haemo Shield® P, Parapleuro Shield® P, ParapleuroShield® P+BE, Rhinicell® FD, Rhini Shield™ TX4, Prefarrow Shield® 9,Prefarrow Strep Shield®, Clostratox® BCD, Clostratox® C, Clostratox®Ultra C 1300, Porcine Ecolizer® 3+C, Porcine Pili Shield™□+C, PorcinePhi Shield™ . . . Porcine Ecolizer® 3, Ery Serum™□□Ery Shield™□□Ery VacOral, Ery Shield™+L5, PanSTAR™ Ery, Erycell™□□Parvo Shield® E, ParvoShield® L5E, Parvo Shield® L5, Parvo Shield®, Para Shield®, PneumoSTARSIV, PneumoSTAR™ Myco, Lepto Shield™ 5, Myco Shield™□□Salmo Shield® 2,Salmo Shield® Live, Amitox Tet™□□C. Perfingens Type A Toxoid (all ofNovartis Animal Health, Basel, Switzerland); Nitro-Sal (Akro); or anyantigen which in included in the compositions described above.Alternatively, when PCV2 antigen is already present in any of thosevaccines, (i) PCV2 antigen, as described herein, is added to any ofthose compostions/antigens, or (ii) the PCV2 antigen present in any ofthose vaccines is replaced by the PCV2 antigen, as described herein.

Formulations

An important aspect of the present invention is the preparation of thecombination vaccine(s). The skilled person knows additional componentswhich may be comprised in said composition (see also Remington'sPharmaceutical Sciences. (1990). 18th ed. Mack Publ., Easton). Theexpert may use known injectable, physiologically acceptable, sterilesolutions. For preparing a ready-to-use solution for parenteralinjection or infusion, aqueous isotonic solutions, such as e.g. salineor corresponding plasma protein solutions, are readily available. Thepharmaceutical compositions may be present as lyophylisates or drypreparations, which can be reconstituted with a known injectablesolution directly before use under sterile conditions, e.g. as a kit ofparts.

In addition, the immunogenic and vaccine compositions of the presentinvention can include one or more veterinary-acceptable carriers. Asused herein, “a veterinary-acceptable carrier” includes any and allsolvents, dispersion media, coatings, adjuvants, stabilizing agents,diluents, preservatives, antibacterial and antifungal agents, isotonicagents, adsorption delaying agents, and the like.

Diluents can include water, saline, dextrose, ethanol, glycerol, and thelike. Isotonic agents can include sodium chloride, dextrose, mannitol,sorbitol, and lactose, among others. Stabilizers include albumin andalkali salts of ethylendiamintetracetic acid, among others.

Preferred adjuvants are those as described above. The immunogeniccompositions can further include one or more other immunomodulatoryagents such as, e. g., interleukins, interferons, or other cytokines.The immunogenic compositions can also include Gentamicin andMerthiolate. While the amounts and concentrations of adjuvants andadditives useful in the context of the present invention can readily bedetermined by the skilled artisan, the present invention contemplatescompositions comprising from about 50 ug to about 2000 ug of adjuvantand preferably about 250 ug/ml dose of the vaccine composition. Inanother preferred embodiment, the present invention contemplates vaccinecompositions comprising from about 1 ug/ml to about 60 ug/ml ofantibiotics, and more preferably less than about 30 ug/ml ofantibiotics.

According to a further embodiment the combination vaccine is firstdehydrated. If the composition is first lyophilized or dehydrated byother methods, then, prior to vaccination, said composition isrehydrated in aqueous (e.g. saline, PBS (phosphate buffered saline)) ornon-aqueous solutions (e.g. oil emulsion (mineral oil, orvegetable/metabolizable oil based/single or double emulsion based),aluminum-based, carbomer based adjuvant).

Dosage and Administration

According to the present invention, an effective amount of a combinationvaccine administered to pigs provides effective immunity againstmicrobiological infections caused by PCV2 and at least one furtherpathogen as listed above. Preferred combinations of antigens for thetreatment and prophylaxis of microbiological diseases in pigs are listedabove.

According to a further embodiment, the combination vaccine isadministered to pigs in one or two doses at an interval of about 2 to 4weeks. For example, the first administration is performed when theanimal is about 2 to 3 weeks to about 8 weeks of age. The secondadministration is performed about 1 to about 4 weeks after the firstadministration of the first vaccination. According to a furtherembodiment, revaccination is performed in an interval of 3 to 12 monthafter administration of the second dose. Administration of subsequentvaccine doses is preferably done on a 6 month to an annual basis. Inanother preferred embodiment, animals vaccinated before the age of about2 to 3 weeks should be revaccinated. Administration of subsequentvaccine doses is preferably done on an annual basis.

The amount of combination vaccine that is effective depends on theingredients of the vaccine and the schedule of administration.Typically, when an inactivated virus or a modified live viruspreparation is used in the combination vaccine, an amount of the vaccinecontaining about 10² to about 10⁹ TCID₅₀ per dose, preferably about 10³to about 10⁸ TCID₅₀ per dose, more preferably, about 10⁴ to about 10⁸TCID₅₀ per dose. In general, inactivated antigen is normally used inhigher amounts than live modivied viruses. Typically, when bacterialantigen is used in the combination vaccine, the vaccine containing anamount of about 10³ to about 10⁹ colony forming units (CFU) per dose,preferably, about 10⁴ to about 10⁸ (CFU) per dose, more preferably about10⁵ to about 10⁶ (CFU) per dose. Sub-unit vaccines are normallyadministered with an antigen inclusion level of at least 0.2 μg antigenper dose, preferably with about 0.2 to about 400 μg/dose, still morepreferably with about 0.3 to about 200 μg/dose, even more preferablywith about 0.35 to about 100 μg/dose, still more preferably with about0.4 to about 50 μg/dose, still more preferably with about 0.45 to about30 μg/dose, still more preferably with about 0.6 to about 15 μg/dose,even more preferably with about 0.75 to about 8 μg/dose, even morepreferably with about 1.0 to about 6 μg/dose, and still more preferablywith about 1.3 to about 3.0 μg/dose. For example, the antigen inclusionlevel of the PCV ORF2 antigen, preferably of the PCV2 ORF2 protein asprovided herewith, contains about 2 μg to about 150 fig, preferablyabout 2 μg to about 60 μg, even more preferably about 2 μg to about 50μg, even more preferably about 2 μg to about 40 μg, even more preferablyabout 2 μg to about 30 μg, even more preferably about 2 μg to about 25μg, even more preferably about 2 μg to about 20 μg, even more preferablyabout 4 μg to about 20 μg, and even more preferably about 4 μg to about16 pig. In the case of combination vaccines that include (37), it ispreferred to use at least 1 to 10 logs, more preferably, 5-10 logs, andmost preferably, 6-8 logs. In the case of combination vaccines thatinclude (41), it is preferred to use at least 1 to 10 logs, morepreferably, 3-10 logs, and most preferably, 5-6 logs.

The composition according to the invention may be applied intradermally,intratracheally, or intravaginally. The composition preferably may beapplied intramuscularly or intranasally. In an animal body, it can proveadvantageous to apply the pharmaceutical compositions as described abovevia an intravenous injection or by direct injection into target tissues.For systemic application, the intravenous, intravascular, intramuscular,intranasal, intraarterial, intraperitoneal, oral, or intrathecal routesare preferred. A more local application can be effected subcutaneously,intradermally, intracutaneously, intracardially, intralobally,intramedullarly, intrapulmonarily or directly in or near the tissue tobe treated (connective-, bone-, muscle-, nerve-, epithelial tissue).Depending on the desired duration and effectiveness of the treatment,the compositions according to the invention may be administered once orseveral times, also intermittently, for instance on a daily basis forseveral days, weeks or months, and in different dosages.

Methods for Treatment

Yet another important embodiment of the invention is a method for theprophylaxis or treatment of diseases caused by PCV2, and one or moreswine pathogenic microorganism(s), wherein a PCV2 antigen, preferably aPCV2 ORF2 protein as provided herewith, and further immunological activecomponents effective for the treatment and/or prophylaxis of theinfection caused by said further swine pathogenic microorganism isadministered to an animal in need thereof at a suitable dosage.According to a further aspect, said PCV2 ORF2 protein, is part of anantigenic composition, as described above. Thus, yet another aspect ofthe present invention relates to a combination vaccine that comprisesany one of the antigenic compositions provided herewith and thatcomprises PCV2 ORF2 protein, and another immunological active componenteffective for the treatment and/or prophylaxis of an infection caused bysaid other swine pathogenic microorganism.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic flow diagram of a preferred construction of PCV2ORF2 recombinant baculovirus; and

FIGS. 2 a and 2 b are each a schematic flow diagram of how to produce acomposition in accordance with the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following examples set forth preferred materials and procedures inaccordance with the present invention. It is to be understood, however,that these examples are provided by way of illustration only, andnothing therein should be deemed a limitation upon the overall scope ofthe invention.

Example 1

This example compares the relative yields of ORF2 using methods of thepresent invention with methods that are known in the prior art. Four1000 mL spinner flasks were each seeded with approximately 1.0×10⁶ Sf+cells/ml in 300 mL of insect serum free media, Excel 420 (JRHBiosciences, Inc., Lenexa, Kans.). The master cell culture is identifiedas SF+(Spodoptera frugiperda) Master Cell Stock, passage 19,Lot#N112-095W. The cells used to generate the SF+Master Cell Stock wereobtained from Protein Sciences Corporation, Inc., Meriden, Conn. The SF+cell line for this example was confined between passages 19 and 59.Other passages will work for purposes of the present invention, but inorder to scale the process up for large scale production, at least 19passages will probably be necessary and passages beyond 59 may have aneffect on expression, although this was not investigated. In moredetail, the initial SF+ cell cultures from liquid nitrogen storage weregrown in Excel 420 media in suspension in sterile spinner flasks withconstant agitation. The cultures were grown in 100 mL to 250 mL spinnerflasks with 25 to 150 mL of Excel 420 serum-free media. When the cellshad multiplied to a cell density of 1.0-8.0×10⁶ cells/mL, they weresplit to new vessels with a planting density of 0.5-1.5×10⁶ cells/mL.Subsequent expansion cultures were grown in spinner flasks up to 36liters in size or in stainless steel bioreactors of up to 300 liters fora period of 2-7 days at 25-29° C.

After seeding, the flasks were incubated at 27° C. for four hours.Subsequently, each flask was seeded with a recombinant baculoviruscontaining the PCV2 ORF2 gene (SEQ ID NO: 4). The recombinantbaculovirus containing the PCV2 ORF2 gene was generated as follows: thePCV2 ORF2 gene from a North American strain of PCV2 was PCR amplified tocontain a 5′ Kozak's sequence (SEQ ID NO: 1) and a 3′ EcoR1 site (SEQ IDNO: 2), cloned into the pGEM-T-Easy vector (Promega, Madison, Wis.).Then, it was subsequently excised and subcloned into the transfer vectorpVL1392 (BD Biosciences Pharmingen, San Diego, Calif.). The subclonedportion is represented herein as SEQ ID NO: 7. The pVL1392 plasmidcontaining the PCV2 ORF2 gene was designated N47-064Y and thenco-transfected with BaculoGold® (BD Biosciences Pharmingen) baculovirusDNA into Sf+ insect cells (Protein Sciences, Meriden, Conn.) to generatethe recombinant baculovirus containing the PCV2 ORF2 gene. The newconstruct is provided herein as SEQ ID NO: 8. The recombinantbaculovirus containing the PCV2 ORF2 gene was plaque-purified and MasterSeed Virus (MSV) was propagated on the SF+ cell line, aliquotted, andstored at −70° C. The MSV was positively identified as PCV2 ORF2baculovirus by PCR-RFLP using baculovirus specific primers. Insect cellsinfected with PCV2 ORF2 baculovirus to generate MSV or Working SeedVirus express PCV2 ORF2 antigen as detected by polyclonal serum ormonoclonal antibodies in an indirect fluorescent antibody assay.Additionally, the identity of the PCV2 ORF2 baculovirus was confirmed byN-terminal amino acid sequencing. The PCV2 ORF2 baculovirus MSV was alsotested for purity in accordance with 9 C.F.R. 113.27 (c), 113.28, and113.55. Each recombinant baculovirus seeded into the spinner flasks hadvarying multiplicities of infection (MOIs). Flask 1 was seeded with 7.52mL of 0.088 MOI seed; flask 2 was seeded with 3.01 mL of 0.36MOI seed;flask 3 was seeded with 1.5 mL of 0.18MOI seed; and flask 4 was seededwith 0.75 mL of 0.09MOI seed. A schematic flow diagram illustrating thebasic steps used to construct a PCV2 ORF2 recombinant baculovirus isprovided herein as FIG. 1.

After being seeded with the baculovirus, the flasks were then incubatedat 27±2° C. for 7 days and were also agitated at 100 rpm during thattime. The flasks used ventilated caps to allow for air flow. Samplesfrom each flask were taken every 24 hours for the next 7 days. Afterextraction, each sample was centrifuged, and both the pellet and thesupernatant were separated and then microfiltered through a 0.45-1.0 μmpore size membrane.

The resulting samples then had the amount of ORF2 present within themquantified via an ELISA assay. The ELISA assay was conducted withcapture antibody Swine anti-PCV2 Pab IgG Prot. G purified (diluted 1:250in PBS) diluted to 1:6000 in 0.05M Carbonate buffer (pH 9.6). 100 μL ofthe antibody was then placed in the wells of the mictrotiter plate,sealed, and incubated overnight at 37° C. The plate was then washedthree times with a wash solution which comprised 0.5 mL of Tween 20(Sigma, St. Louis, Mo.), 100 mL of 10× D-PBS (Gibco Invitrogen,Carlsbad, Calif.) and 899.5 mL of distilled water. Subsequently, 250 μLof a blocking solution (5 g Carnation Non-fat dry milk (Nestle,Glendale, Calif.) in 10 mL of D-PBS QS to 100 mL with distilled water)was added to each of the wells. The next step was to wash the test plateand then add pre-diluted antigen. The pre-diluted antigen was producedby adding 200 μL of diluent solution (0.5 mL Tween 20 in 999.5 mL D-PBS)to each of the wells on a dilution plate. The sample was then diluted ata 1:240 ratio and a 1:480 ratio, and 100 μL of each of these dilutedsamples was then added to one of the top wells on the dilution plate(i.e. one top well received 100 μL of the 1:240 dilution and the otherreceived 100 μL of the 1:480 dilution). Serial dilutions were then donefor the remainder of the plate by removing 100 μL form each successivewell and transferring it to the next well on the plate. Each well wasmixed prior to doing the next transfer. The test plate washing includedwashing the plate three times with the wash buffer. The plate was thensealed and incubated for an hour at 37° C. before being washed threemore times with the wash buffer. The detection antibody used wasmonoclonal antibody to PCV ORF2. It was diluted to 1:300 in diluentsolution, and 100 μL of the diluted detection antibody was then added tothe wells. The plate was then sealed and incubated for an hour at 37° C.before being washed three times with the wash buffer. Conjugate diluentwas then prepared by adding normal rabbit serum (Jackson Immunoresearch,West Grove, Pa.) to the diluent solution to 1% concentration. Conjugateantibody Goat anti-mouse (H+1)-HRP (Jackson Immunoresearch) was dilutedin the conjugate diluent to 1:10,000. 100 μL of the diluted conjugateantibody was then added to each of the wells. The plate was then sealedand incubated for 45 minutes at 37° C. before being washed three timeswith the wash buffer. 100 μL of substrate (TMB Peroxidase Substrate,Kirkgaard and Perry Laboratories (KPL), Gaithersberg, Md.), mixed withan equal volume of Peroxidase Substrate B (KPL) was added to each of thewells. The plate was incubated at room temperature for 15 minutes. 100μL of 1N HCL solution was then added to all of the wells to stop thereaction. The plate was then run through an ELISA reader. The results ofthis assay are provided in Table 1 below:

TABLE 1 ORF2 in ORF2 in Day Flask pellet (μg) supernatant (μg) 3 1 47.5312 3 2 57.46 22 3 3 53.44 14 3 4 58.64 12 4 1 43.01 44 4 2 65.61 62 4 370.56 32 4 4 64.97 24 5 1 31.74 100 5 2 34.93 142 5 3 47.84 90 5 4 55.1486 6 1 14.7 158 6 2 18.13 182 6 3 34.78 140 6 4 36.88 146 7 1 6.54 176 72 12.09 190 7 3 15.84 158 7 4 15.19 152

These results indicate that when the incubation time is extended,expression of ORF2 into the supernatant of the centrifuged cells andmedia is greater than expression in the pellet of the centrifuged cellsand media. Accordingly, allowing the ORF2 expression to proceed for atleast 5 days and recovering it in the supernate rather than allowingexpression to proceed for less than 5 days and recovering ORF2 from thecells, provides a great increase in ORF2 yields, and a significantimprovement over prior methods.

Example 2

This example provides data as to the efficacy of the invention claimedherein. A 1000 mL spinner flask was seeded with approximately 1.0×10⁶Sf+cells/ml in 300 mL of Excel 420 media. The flask was then incubated at27° C. and agitated at 100 rpm. Subsequently, the flask was seeded with10 mL of PCV2 ORF2/Bac p+6 (the recombinant baculovirus containing thePCV2 ORF2 gene passaged 6 additional times in the Sf9 insect cells)virus seed with a 0.1 MOI after 24 hours of incubation.

The flask was then incubated at 27° C. for a total of 6 days. Afterincubation, the flask was then centrifuged and three samples of theresulting supernatant were harvested and inactivated. The supernatantwas inactivated by bringing its temperature to 37±2° C. To the firstsample, a 0.4M solution of 2-bromoethyleneamine hydrobromide which hadbeen cyclized to 0.2M binary ethlylenimine (BEI) in 0.3N NaOH is addedto the supernatant to give a final concentration of BEI of 5 mM. To thesecond sample, 10 mM BEI was added to the supernatant. To the thirdsample, no BEI was added to the supernatant. The samples were thenstirred continuously for 48 hrs. A 1.0 M sodium thiosulfate solution togive a final minimum concentration of 5 mM was added to neutralize anyresidual BEI. The quantity of ORF2 in each sample was then quantifiedusing the same ELISA assay procedure as described in Example 1. Theresults of this may be seen in Table 2 below:

TABLE 2 Sample ORF2 in supernatant (μg) 1 78.71 2 68.75 3 83.33

This example demonstrates that neutralization with BEI does not removeor degrade significant amounts of the recombinant PCV2 ORF2 proteinproduct. This is evidenced by the fact that there is no large loss ofORF2 in the supernatant from the BEI or elevated temperatures. Those ofskill in the art will recognize that the recovered ORF2 is a stableprotein product.

Example 3

This example demonstrates that the present invention is scalable fromsmall scale production of recombinant PCV2 ORF2 to large scaleproduction of recombinant PCV2 ORF2. 5.0×10⁵ cells/ml of SF+ cells/ml in7000 mL of ExCell 420 media was planted in a 20000 mL ApplikonBioreactor. The media and cells were then incubated at 27° C. andagitated at 100RPM for the next 68 hours. At the 68^(th) hour, 41.3 mLof PCV2 ORF2 Baculovirus MSV+3 was added to 7000 mL of ExCell 420medium. The resultant mixture was then added to the bioreactor. For thenext seven days, the mixture was incubated at 27° C. and agitated at100RPM. Samples from the bioreactor were extracted every 24 hoursbeginning at day 4, post-infection, and each sample was centrifuged. Thesupernatant of the samples were preserved and the amount of ORF2 wasthen quantified using SDS-PAGE densitometry. The results of this can beseen in Table 3 below:

TABLE 3 Day after infection: ORF2 in supernatant (μg/mL) 4 29.33 5 41.336 31.33 7 60.67

Example 4

This example tests the efficacy of seven PCV2 candidate vaccines andfurther defines efficacy parameters following exposure to a virulentstrain of PCV2. One hundred and eight (108) cesarean derived colostrumdeprived (CDCD) piglets, 9-14 days of age, were randomly divided into 9groups of equal size. Table 4 sets forth the General Study Design forthis Example.

TABLE 4 General Study Design Challenged KLH/ICF with No. Day of A on DayVirulent Necropsy Of Treat- 21 and PCV2 on on Group Pigs Treatment mentDay 27 Day 24 Day 49 1 12 PCV2 Vaccine 0 + + + No. 1— (vORF2 16 μg) 2 12PCV2 Vaccine 0 + + + No. 2— (vORF2 8 μg) 3 12 PCV2 Vaccine 0 + + + No.3— (vORF2 4 μg) 4 12 PCV2 Vaccine 0 + + + No. 4— (rORF2 16 μg) 5 12 PCV2Vaccine 0 + + + No. 5— (rORF2 8 μg) 6 12 PCV2 Vaccine 0 + + + No. 6—(rORF2 4 μg) 7 12 PCV2 Vaccine 0 + + + No. 7— (Killed whole cell virus)8 12 None— N/A + + + Challenge Controls 9 12 None—Strict N/A + − +Negative Control Group vORF2 = isolated viral ORF2; rORF2 = recombinantbaculovirus expressed ORF2; killed whole cell virus = PCV2 virus grownin suitable cell culture

Seven of the groups (Groups 1-7) received doses of PCV2 ORF2polypeptide, one of the groups acted as a challenge control and receivedno PCV2 ORF2, and another group acted as the strict negative controlgroup and also received no PCV2 ORF2. On Day 0, Groups 1 through 7 weretreated with assigned vaccines. Piglets in Group 7 were given a boostertreatment on Day 14. Piglets were observed for adverse events andinjection site reactions following vaccination and on Day 19, pigletswere moved to the second study site. At the second study site, Groups1-8 were group housed in one building while Group 9 was housed in aseparate building. All pigs received keyhole limpet hemocyanin(KLH)/incomplete Freund's adjuvant (ICFA) on Days 21 and 27 and on Day24, Groups 1-8 were challenged with a virulent PCV2.

Pre- and post-challenge, blood samples were collected for PCV2 serology.Post-challenge, body weight data for determination of average dailyweight gain (ADWG), and clinical symptoms, as well as nasal swab samplesto determine nasal shedding of PCV2, were collected. On Day 49, allsurviving pigs were necropsied, lungs were scored for lesions, andselected tissues were preserved in formalin for Immunohistochemistry(IHC) testing at a later date.

Materials and Methods

This was a partially blinded vaccination-challenge feasibility studyconducted in CDCD pigs, 9 to 14 days of age on Day 0. To be included inthe study, PCV2 IFA titers of sows were <1:1000. Additionally, theserologic status of sows were from a known PRRS-negative herd.Twenty-eight (28) sows were tested for PCV2 serological status. Fourteen(14) sows had a PCV2 titer of ≦1000 and were transferred to the firststudy site. One hundred ten (110) piglets were delivered by cesareansection surgeries and were available for this study on Day −4. On Day−3, 108 CDCD pigs at the first study site were weighed, identified withear tags, blocked by weight and randomly assigned to 1 of 9 groups, asset forth above in table 4. If any test animal meeting the inclusioncriteria was enrolled in the study and was later excluded for anyreason, the Investigator and Monitor consulted in order to determine theuse of data collected from the animal in the final analysis. The date ofwhich enrolled piglets were excluded and the reason for exclusion wasdocumented. Initially, no sows were excluded. A total of 108 of anavailable 110 pigs were randomly assigned to one of 9 groups on Day −3.The two smallest pigs (No. 17 and 19) were not assigned to a group andwere available as extras, if needed. During the course of the study,several animals were removed. Pig 82 (Group 9) on Day −1, Pig No. 56(Group 6) on Day 3, Pig No. 53 (Group 9) on Day 4, Pig No. 28 (Group 8)on Day 8, Pig No. 69 (Group 8) on Day 7, and Pig No. 93 (Group 4) on Day9, were each found dead prior to challenge. These six pigs were notincluded in the final study results. Pig no 17 (one of the extra pigs)was assigned to Group 9. The remaining extra pig, No. 19, was excludedfrom the study.

The formulations given to each of the groups were as follows: Group 1was designed to administer 1 ml of viral ORF2 (vORF2) containing 16 μgORF2/ml. This was done by mixing 10.24 ml of viral ORF2 (256 μg/25μg/ml=10.24 ml vORF2) with 3.2 ml of 0.5% Carbopol and 2.56 ml ofphosphate buffered saline at a pH of 7.4. This produced 16 ml offormulation for group 1. Group 2 was designed to administer 1 ml ofvORF2 containing 8 μg vORF2/ml. This was done by mixing 5.12 ml of vORF2(128 μg/25 μg/ml=5.12 ml vORF2) with 3.2 ml of 0.5% Carbopol and 7.68 mlof phosphate buffered saline at a pH of 7.4. This produced 16 ml offormulation for group 2. Group 3 was designed to administer 1 ml ofvORF2 containing 4 μg vORF2/ml. This was done by mixing 2.56 ml of vORF2(64 μg/25 μg/ml=2.56 ml vORF2) with 3.2 ml of 0.5% Carbopol and 10.24 mlof phosphate buffered saline at a pH of 7.4. This produced 16 ml offormulation for group 3. Group 4 was designed to administer 1 ml ofrecombinant ORF2 (rORF2) containing 16 μg rORF2/ml. This was done bymixing 2.23 ml of rORF2 (512 μg/230 μg/ml=2.23 ml rORF2) with 6.4 ml of0.5% Carbopol and 23.37 ml of phosphate buffered saline at a pH of 7.4.This produced 32 ml of formulation for group 4. Group 5 was designed toadminister 1 ml of rORF2 containing 8 μg rORF2/ml. This was done bymixing 1.11 ml of rORF2 (256 μg/230 μg/ml=1.11 ml rORF2) with 6.4 ml of0.5% Carbopol and 24.49 ml of phosphate buffered saline at a pH of 7.4.This produced 32 ml of formulation for group 5. Group 6 was designed toadminister 1 ml of rORF2 containing 8 μg rORF2/ml. This was done bymixing 0.56 ml of rORF2 (128 μg/230 μg/ml=0.56 ml rORF2) with 6.4 ml of0.5% Carbopol and 25.04 ml of phosphate buffered saline at a pH of 7.4.This produced 32 ml of formulation for group 6. Group 7 was designed toadminister 2 ml of PCV2 whole killed cell vaccine (PCV2 KV) containingthe MAX PCV2 KV. This was done by mixing 56 ml of PCV2 KV with 14 ml of0.5% Carbopol. This produced 70 ml of formulation for group 7 Finallygroup 8 was designed to administer KLH at 0.5 μg/ml or 1.0 μg/ml per 2ml dose. This was done by mixing 40.71 ml KLH (7.0 μg protein/ml at 0.5μg/ml=570 ml (7.0 μg/ml)(x)=(0.5)(570 ml)), 244.29 ml phosphate bufferedsaline at a pH of 7.4, and 285 ml Freunds adjuvant. Table 5 describesthe time frames for the key activities of this Example.

TABLE 5 Study Activities Study Day Study Activity −4, 0 to 49 Generalobservations for overall health and clinical symptoms −3 Weighed;Randomized to groups; Collected blood samples from all pigs 0 Healthexamination; Administered IVP Nos. 1-7 to Groups 1-7, respectively 0-7Observed pigs for injection site reactions 14 Boostered Group 7 withPCV2 Vaccine No. 7; Blood samples from all pigs 14-21 Observed Group 7for injection site reactions 16-19 Treated all pigs with antibiotics(data missing) 19 Pigs transported from the first test site to a secondtest site 21 Treated Groups 1-9 with KLH/ICFA 24 Collected blood andnasal swab samples from all pigs; Weighed all pigs; Challenged Groups1-8 with PCV2 challenge material 25, 27, Collected nasal swab samplesfrom all pigs 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 27 Treated Groups1-9 with KLH/ICFA 31 Collected blood samples from all pigs 49 Collectedblood and nasal swab samples from all pigs; Weighed all pigs; Necropsyall pigs; Gross lesions noted with emphasis placed on icterus andgastric ulcers; Lungs evaluated for lesions; Fresh and formalin fixedtissue samples saved; In-life phase of the study completed

Following completion of the in-life phase of the study, formalin fixedtissues were examined by Immunohistochemistry (IHC) for detection ofPCV2 antigen by a pathologist, blood samples were evaluated for PCV2serology, nasal swab samples were evaluated for PCV2 shedding, andaverage daily weight gain (ADWG) was determined from Day 24 to Day 49.

Animals were housed at the first study site in individual cages in fiverooms from birth to approximately 11 days of age (approximately Day 0 ofthe study). Each room was identical in layout and consisted of stackedindividual stainless steel cages with heated and filtered air suppliedseparately to each isolation unit. Each room had separate heat andventilation, thereby preventing cross-contamination of air betweenrooms. Animals were housed in two different buildings at the secondstudy site. Group 9 (The Strict negative control group) was housedseparately in a converted finisher building and Groups 1-8 were housedin converted nursery building. Each group was housed in a separate pen(11-12 pigs per pen) and each pen provided approximately 3.0 square feetper pig. Each pen was on an elevated deck with plastic slatted floors. Apit below the pens served as a holding tank for excrement and waste.Each building had its own separate heating and ventilation systems, withlittle likelihood of cross-contamination of air between buildings.

At the first study site, piglets were fed a specially formulated milkration from birth to approximately 3 weeks of age. All piglets wereconsuming solid, special mixed ration by Day 19 (approximately 4½ weeksof age). At the second study site, all piglets were fed a customnon-medicated commercial mix ration appropriate for their age andweight, ad libitum. Water at both study sites was also available adlibitum.

All test pigs were treated with Vitamin E on Day −2, with ironinjections on Day −1 and with NAXCEL® (1.0 mL, IM, in alternating hams)on Days 16, 17, 18 and 19. In addition, Pig No. 52 (Group 9) was treatedwith an iron injection on Day 3, Pig 45 (Group 6) was treated with aniron injection on Day 11, Pig No. 69 (Group 8) was treated with NAXCEL®on Day 6, Pig No. 74 (Group 3) was treated with dexamethazone andpenicillin on Day 14, and Pig No. 51 (Group 1) was treated withdexamethazone and penicillin on Day 13 and with NAXCEL® on Day 14 forvarious health reasons.

While at both study sites, pigs were under veterinary care. Animalhealth examinations were conducted on Day 0 and were recorded on theHealth Examination Record Form. All animals were in good health andnutritional status before vaccination as determined by observation onDay 0. All test animals were observed to be in good health andnutritional status prior to challenge. Carcasses and tissues weredisposed of by rendering. Final disposition of study animals was recordson the Animal Disposition Record.

On Day 0, pigs assigned to Groups 1-6 received 1.0 mL of PCV2 Vaccines1-6, respectively, IM in the left neck region using a sterile 3.0 mLLuer-lock syringe and a sterile 20 g×½″ needle. Pigs assigned to Group 7received 2.0 mL of PCV2 Vaccine No. 7 IM in the left neck region using asterile 3.0 mL Luer-lock syringe and a sterile 20 g×½″ needle. On Day14, pigs assigned to Group 7 received 2.0 mL of PCV2 Vaccine No. 7 IM inthe right neck region using a sterile 3.0 mL Luer-lock syringe and asterile 20 g×½″ needle.

On Day 21 all test pigs received 2.0 mL of KLH/ICFA IM in the right hamregion using a sterile 3.0 mL Luer-lock syringe and a sterile 20 g×1″needle. On Day 27 all test pigs received 2.0 mL of KLH/ICFA in the leftham region using a sterile 3.0 mL Luer-lock syringe and a sterile 20g×1″ needle.

On Day 24, pigs assigned to Groups 1-8 received 1.0 mL of PCV2 ISUVDLchallenge material (5.11 log₁₀ TCID₅₀/mL) IM in the left neck regionusing a sterile 3.0 mL Luer-lock syringe and a sterile 20 g×1″ needle.An additional 1.0 mL of the same material was administered IN to eachpig (0.5 mL per nostril) using a sterile 3.0 mL Luer-lock syringe andnasal canula.

Test pigs were observed daily for overall health and adverse events onDay −4 and from Day 0 to Day 19. Observations were recorded on theClinical Observation Record. All test pigs were observed from Day 0 toDay 7, and Group 7 was further observed from Day 14 to 21, for injectionsite reactions. Average daily weight gain was determined by weighingeach pig on a calibrated scale on Days −3, 24 and 49, or on the day thata pig was found dead after challenge. Body weights were recorded on theBody Weight Form. Day −3 body weights were utilized to block pigs priorto randomization. Day 24 and Day 49 weight data was utilized todetermine the average daily weight gain (ADWG) for each pig during thesetime points. For pigs that died after challenge and before Day 49, theADWG was adjusted to represent the ADWG from Day 24 to the day of death.

In order to determine PCV2 serology, venous whole blood was collectedfrom each piglet from the orbital venous sinus on Days −3 and 14. Foreach piglet, blood was collected from the orbital venous sinus byinserting a sterile capillary tube into the medial canthus of one of theeyes and draining approximately 3.0 mL of whole blood into a 4.0 mLSerum Separator Tube (SST). On Days 24, 31, and 49, venous whole bloodfrom each pig was collected from the anterior vena cava using a sterile18 g×1½″ Vacutainer needle (Becton Dickinson and Company, FranklinLakes, N.J.), a Vacutainer needle holder and a 13 mL SST. Bloodcollections at each time point were recorded on the Sample CollectionRecord. Blood in each SST was allowed to clot, each SST was then spundown and the serum harvested. Harvested serum was transferred to asterile snap tube and stored at −70±10° C. until tested at a later date.Serum samples were tested for the presence of PCV2 antibodies byBIVI-R&D personnel.

Pigs were observed once daily from Day 20 to Day 49 for clinicalsymptoms and clinical observations were recorded on the ClinicalObservation Record.

To test for PCV2 nasal shedding, on Days 24, 25, and then every otherodd numbered study day up to and including Day 49, a sterile dacron swabwas inserted intra nasally into either the left or right nostril of eachpig (one swab per pig) as aseptically as possible, swished around for afew seconds and then removed. Each swab was then placed into a singlesterile snap-cap tube containing 1.0 mL of EMEM media with 2% IFBS, 500units/mL of Penicillin, 500 μg/mL of Streptomycin and 2.5 μg/mL ofFungizone. The swab was broken off in the tube, and the snap tube wassealed and appropriately labeled with animal number, study number, dateof collection, study day and “nasal swab.” Sealed snap tubes were storedat −40±10° C. until transported overnight on ice to BIVI-St. Joseph.Nasal swab collections were recorded on the Nasal Swab Sample CollectionForm. BIVI-R&D conducted quantitative virus isolation (VI) testing forPCV2 on nasal swab samples. The results were expressed in log₁₀ values.A value of 1.3 logs or less was considered negative and any valuegreater than 1.3 logs was considered positive.

Pigs that died (Nos. 28, 52, 56, 69, 82, and 93) at the first study sitewere necropsied to the level necessary to determine a diagnosis. Grosslesions were recorded and no tissues were retained from these pigs. Atthe second study site, pigs that died prior to Day 49 (Nos. 45, 23, 58,35), pigs found dead on Day 49 prior to euthanasia (Nos. 2, 43) and pigseuthanized on Day 49 were necropsied. Any gross lesions were noted andthe percentages of lung lobes with lesions were recorded on the NecropsyReport Form.

From each of the 103 pigs necropsied at the second study site, a tissuesample of tonsil, lung, heart, liver, mesenteric lymph node, kidney andinguinal lymph node was placed into a single container with buffered 10%formalin; while another tissue sample from the same aforementionedorgans was placed into a Whirl-pak (M-Tech Diagnostics Ltd., Thelwall,UK) and each Whirl-pak was placed on ice. Each container was properlylabeled. Sample collections were recorded on the Necropsy Report Form.Afterwards, formalin-fixed tissue samples and a Diagnostic Request Formwere submitted for IHC testing. IHC testing was conducted in accordancewith standard ISU laboratory procedures for receiving samples, sampleand slide preparation, and staining techniques. Fresh tissues inWhirl-paks were shipped with ice packs to the Study Monitor for storage(−70°±10° C.) and possible future use. Formalin-fixed tissues wereexamined by a pathologist for detection of PCV2 by IHC and scored usingthe following scoring system: 0=None; 1=Scant positive staining, fewsites; 2=Moderate positive staining, multiple sites; and 3=Abundantpositive staining, diffuse throughout the tissue. Due to the fact thatthe pathologist could not positively differentiate inguinal LN frommesenteric LN, results for these tissues were simply labeled as LymphNode and the score given the highest score for each of the two tissuesper animal.

Results

Results for this example are given below. It is noted that one pig fromGroup 9 died before Day 0, and 5 more pigs died post-vaccination (1 pigfrom Group 4; 1 pig from Group 6; 2 pigs from Group 8; and 1 pig fromGroup 9). Post-mortem examination indicated all six died due tounderlying infections that were not associated with vaccination or PMWS.Additionally, no adverse events or injection site reactions were notedwith any groups.

Average daily weight gain (ADWG) results are presented below in Table 6.Group 9, the strict negative control group, had the highest ADWG(1.06±0.17 lbs/day), followed by Group 5 (0.94±0.22 lbs/day), whichreceived one dose of 8 μg of rORF2. Group 3, which received one dose of4 μg of vORF2, had the lowest ADWG (0.49±0.21 lbs/day), followed byGroup 7 (0.50±0.15 lbs/day), which received 2 doses of killed vaccine.

TABLE 6 Summary of Group Average Daily Weight Gain (ADWG) ADWG-lbs/day(Day 24 to Day 49) or Group Treatment N adjusted for pigs dead beforeDay 29 1 vORF2-16 μg (1 dose) 12 0.87 ± 0.29 lbs/day 2 vORF2-8 μg (1dose) 12 0.70 ± 0.32 lbs/day 3 vORF2-4 μg (1 dose) 12 0.49 ± 0.21lbs/day 4 rORF2-16 μg (1 dose) 11 0.84 ± 0.30 lbs/day 5 rORF2-8 μg (1dose) 12 0.94 ± 0.22 lbs/day 6 rORF2-4 μg (1 dose) 11 0.72 ± 0.25lbs/day 7 KV (2 doses) 12 0.50 ± 0.15 lbs/day 8 Challenge Controls 100.76 ± 0.19 lbs/day 9 Strict Negative Controls 11 1.06 ± 0.17 lbs/dayvORF2 = isolated viral ORF2; rORF2 = recombinant baculovirus expressedORF2; killed whole cell virus = PCV2 virus grown in suitable cellculture

PCV2 serology results are presented below in Table 7. All nine groupswere seronegative for PCV2 on Day −3. On Day 14, Groups receiving vORF2vaccines had the highest titers, which ranged from 187.5 to 529.2. Pigsreceiving killed viral vaccine had the next highest titers, followed bythe groups receiving rORF2 vaccines. Groups 8 and 9 remainedseronegative at this time. On Day 24 and Day 31, pigs receiving vORF2vaccines continued to demonstrate a strong serological response,followed closely by the group that received two doses of a killed viralvaccine. Pigs receiving rORF2 vaccines were slower to respondserologically and Groups 8 and 9 continued to remain seronegative. OnDay 49, pigs receiving vORF2 vaccine, 2 doses of the killed viralvaccine and the lowest dose of rORF2 demonstrated the strongestserological responses. Pigs receiving 16 μg and 8 μg of rORF2 vaccineshad slightly higher IFA titers than challenge controls. Group 9 on Day49 demonstrated a strong serological response.

TABLE 7 Summary of Group PCV2 IFA Titers AVERAGE IFA TITER Day Day DayDay Day Group Treatment −3 14 24 31** 49*** 1 vORF2—16 μg 50.0 529.24400.0 7866.7 11054.5 (1 dose) 2 vORF2—8 μg 50.0 500.0 3466.7 6800.010181.8 (1 dose) 3 vORF2—4 μg 50.0 187.5 1133.3 5733.3 9333.3 (1 dose) 4rORF2—16 μg 50.0 95.5 1550.0 3090.9 8000.0 (1 dose) 5 rORF2—8 μg 50.075.0 887.5 2266.7 7416.7 (1 dose) 6 rORF2—4 μg 50.0 50.0 550.0 3118.210570.0 (1 dose) 7 KV (2 doses) 50.0 204.2 3087.5 4620.8 8680.0 8Challenge 50.0 55.0 50.0 50.0 5433.3 Controls 9 Strict Negative 50.059.1 59.1 54.5 6136.4 Controls vORF2 = isolated viral ORF2; rORF2 =recombinant baculovirus expressed ORF2; killed whole cell virus = PCV2virus grown in suitable cell culture *For calculation purposes, a ≦100IFA titer was designated as a titer of “50”; a ≧6400 IFA titer wasdesignated as a titer of “12,800”. **Day of Challenge ***Day of Necropsy

The results from the post-challenge clinical observations are presentedbelow in Table 8. This summary of results includes observations forAbnormal Behavior, Abnormal Respiration, Cough and Diarrhea. Table 9includes the results from the Summary of Group Overall Incidence ofClinical Symptoms and Table 10 includes results from the Summary ofGroup Mortality Rates Post-challenge. The most common clinical symptomnoted in this study was abnormal behavior, which was scored as mild tosevere lethargy. Pigs receiving the 2 lower doses of vORF2, pigsreceiving 16 μg of rORF2 and pigs receiving 2 doses of KV vaccine hadincidence rates of ≧27.3%. Pigs receiving 8 μg of rORF2 and the strictnegative control group had no abnormal behavior. None of the pigs inthis study demonstrated any abnormal respiration. Coughing was notedfrequently in all groups (0 to 25%), as was diarrhea (0-20%). None ofthe clinical symptoms noted were pathognomic for PMWS.

The overall incidence of clinical symptoms varied between groups. Groupsreceiving any of the vORF2 vaccines, the group receiving 16 μg of rORF2,the group receiving 2 doses of KV vaccine and the challenge controlgroup had the highest incidence of overall clinical symptoms (≧36.4%).The strict negative control group, the group receiving 8 μg of rORF2 andthe group receiving 4 μg of rORF2 had overall incidence rates ofclinical symptoms of 0%, 8.3% and 9.1%, respectively.

Overall mortality rates between groups varied as well. The groupreceiving 2 doses of KV vaccine had the highest mortality rate (16.7%);while groups that received 4 μg of vORF2, 16 μg of rORF2, or 8 μg ofrORF2 and the strict negative control group all had 0% mortality rates.

TABLE 8 Summary of Group Observations for Abnormal Behavior, AbnormalRespiration, Cough, and Diarrhea Abnormal Abnormal Group Treatment NBehavior¹ Behavior² Cough³ Diarrhea⁴ 1 vORF2—16 μg 12 2/12 0/12 3/122/12 (1 dose) (16.7%) (0%) (25%) (16/7%) 2 vORF2—8 μg 12 4/12 0/12 1/121/12 (1 dose) (33.3%) (0%) (8.3%) (8.3%) 3 vORF2—4 μg 12 8/12 0/12 2/121/12 (1 dose) (66.7%) (0%) (16.7%) (8.3%) 4 rORF2—16 μg 11 3/11 0/110/11 2/11 (1 dose) (27.3%) (0%) (0%) (18.2%) 5 rORF2—8 μg 12 0/12 0/121/12 0/12 (1 dose) (0%) (0%) (8.3%) (0%) 6 rORF2—4 μg 11 1/11 0/11 0/110/12 (1 dose) (9.1%) (0%) (0%) (0%) 7 KV (2 doses) 12 7/12 0/12 0/121/12 (58.3) (0%) (0%) (8.3%) 8 Challenge 10 1/10 0/10 2/10 2/10 Controls(10%) (0%) (20%) (20%) 9 Strict Negative 11 0/11 0/11 0/11 0/11 Controls(0%) (0%) (0%) (0%) vORF2 = isolated viral ORF2; rORF2 = recombinantbaculovirus expressed ORF2; killed whole cell virus = PCV2 virus grownin suitable cell culture ¹Total number of pigs in each group thatdemonstrated any abnormal behavior for at least one day ²Total number ofpigs in each group that demonstrated any abnormal respiration for atleast one day ³Total number of pigs in each group that demonstrated acough for at least one day ⁴Total number of pigs in each group thatdemonstrated diarrhea for at least one day

TABLE 9 Summary of Group Overall Incidence of Clinical SymptomsIncidence of pigs with Incidence Group Treatment N Clinical Symptoms¹Rate 1 vORF2-16 μg (1 dose) 12 5 41.7% 2 vORF2-8 μg (1 dose) 12 5 41.7%3 vORF2-4 μg (1 dose) 12 8 66.7% 4 rORF2-16 μg (1 dose) 11 4 36.4% 5rORF2-8 μg (1 dose) 12 1  8.3% 6 rORF2-4 μg (1 dose) 11 1  9.1% 7 KV (2doses) 12 7 58.3% 8 Challenge Controls 10 4   40% 9 Strict NegativeControls 11 0   0% vORF2 = isolated viral ORF2; rORF2 = recombinantbaculovirus expressed ORF2; killed whole cell virus = PCV2 virus grownin suitable cell culture ¹Total number of pigs in each group thatdemonstrated any clinical symptom for at least one day

TABLE 10 Summary of Group Mortality Rates Post-challenge Dead Post-Mortality Group Treatment N challenge Rate 1 vORF2-16 μg (1 dose) 12 1 8.3% 2 vORF2-8 μg (1 dose) 12 1  8.3% 3 vORF2-4 μg (1 dose) 12 0   0% 4rORF2-16 μg (1 dose) 11 0   0% 5 rORF2-8 μg (1 dose) 12 0   0% 6 rORF2-4μg (1 dose) 11 1  9.1% 7 KV (2 doses) 12 2 16.7% 8 Challenge Controls 101   10% 9 Strict Negative Controls 11 0   0% vORF2 = isolated viralORF2; rORF2 = recombinant baculovirus expressed ORF2; killed whole cellvirus = PCV2 virus grown in suitable cell culture

PCV2 nasal shedding results are presented below in Table 11. Followingchallenge on Day 24, 1 pig in Group 7 began shedding PCV2 on Day 27.None of the other groups experienced shedding until Day 33. The bulk ofnasal shedding was noted from Day 35 to Day 45. Groups receiving any ofthe three vORF2 vaccines and groups receiving either 4 or 8 μg of rORF2had the lowest incidence of nasal shedding of PCV2 (<9.1%). Thechallenge control group (Group 8) had the highest shedding rate (80%),followed by the strict negative control group (Group 9), which had anincidence rate of 63.6%.

TABLE 11 Summary of Group Incidence of Nasal Shedding of PCV2 No. Ofpigs that shed for Incidence Group Treatment N at least one day Rate 1vORF2-16 μg (1 dose) 12 1  8.3% 2 vORF2-8 μg (1 dose) 12 1  8.3% 3vORF2-4 μg (1 dose) 12 1  8.3% 4 rORF2-16 μg (1 dose) 11 2 18.2% 5rORF2-8 μg (1 dose) 12 1  8.3% 6 rORF2-4 μg (1 dose) 11 1  9.1% 7 KV (2doses) 12 5 41.7% 8 Challenge Controls 10 8   80% 9 Strict NegativeControls 11 7 63.6% vORF2 = isolated viral ORF2; rORF2 = recombinantbaculovirus expressed ORF2; killed whole cell virus = PCV2 virus grownin suitable cell culture

The Summary of Group Incidence of Icterus, Group Incidence of GastricUlcers, Group Mean Lung Lesion Scores, and Group Incidence of LungLesions are shown below in Table 12. Six pigs died at the first testsite during the post-vaccination phase of the study (Group 4, N=1; Group6, N=1; Group 8, N=2; Group 9, N=2). Four out of six pigs had fibrinouslesions in one or more body cavities, one pig (Group 6) had lesionsconsistent with clostridial disease, and one pig (Group 9) had no grosslesions. None of the pigs that died during the post-vaccination phasedof the study had lesions consistent with PMWS.

Pigs that died post-challenge and pigs euthanized on Day 49 werenecropsied. At necropsy, icterus and gastric ulcers were not present inany group. With regard to mean % lung lesions, Group 9 had lowest mean %lung lesions (0%), followed by Group 1 with 0.40±0.50% and Group 5 with0.68±1.15%. Groups 2, 3, 7 and 8 had the highest mean % lung lesions(>7.27%). Each of these four groups contained one pig with % lunglesions >71.5%, which skewed the results higher for these four groups.With the exception of Group 9 with 0% lung lesions noted, the remaining8 groups had <36% lung lesions. Almost all lung lesions noted weredescribed as red/purple and consolidated.

TABLE 12 Summary of Group Incidence of Icterus, Group Incidence ofGastric Ulcers, Group Mean % Lung Lesion Scores, and Group Incidence ofLung Lesions Noted Incidence Mean % of Lung Gastric Lung Lesions GroupTreatment Icterus Ulcers Lesions Noted 1 vORF2-16 μg 0/12 (0%) 0/12 0.40± 0.50% 10/12 (1 dose) (0%) (83%) 2 vORF2-8 μg 0/12 (0%) 0/12 7.41 ±20.2% 10/12 (1 dose) (0%) (83%) 3 vORF2-4 μg 0/12 (0%) 0/12 9.20 ± 20.9%10/12 (1 dose) (0%) (83%) 4 rORF2-16 μg 0/11 (0%) 0/11  1.5 ± 4.74% 4/11(1 dose) (0%) (36%) 5 rORF2-8 μg 0/12 (0%) 0/12 0.68 ± 1.15% 9/12 (1dose) (0%) (75%) 6 rORF2-4 μg 0/11 (0%) 0/11 2.95 ± 5.12% 7/11 (1 dose)(0%) (64%) 7 KV 0/12 (0%) 0/12 7.27 ± 22.9% 9/12 (2 doses) (0%) (75%) 8Challenge 0/10 (0%) 0/10 9.88 ± 29.2% 8/10 Controls (0%) (80%) 9 StrictNegative 0/11 (0%) 0/11 0/11 0/11 Controls (0%) (0%) (0%) vORF2 =isolated viral ORF2; rORF2 = recombinant baculovirus expressed ORF2; KVor killed whole cell virus = PCV2 virus grown in suitable cell culture

The Summary of Group IHC Positive Incidence Results are shown in Table13. Group 1 (vORF2-16 μg) and Group 5 (rORF2-8 μg) had the lowest rateof IHC positive results (16.7%). Group 8 (Challenge Controls) and Group9 (Strict Negative Controls) had the highest rate of IHC positiveresults, 90% and 90.9%, respectively.

TABLE 13 Summary of Group IHC Positive Incidence Rate No. Of pigs thathad at least one tissue positive Incidence Group Treatment N for PCV2Rate 1 vORF2-16 μg (1 dose) 12 2 16.7% 2 vORF2-8 μg (1 dose) 12 3 25.0%3 vORF2-4 μg (1 dose) 12 8 66.7% 4 rORF2-16 μg (1 dose) 11 4 36.3% 5rORF2-8 μg (1 dose) 12 2 16.7% 6 rORF2-4 μg (1 dose) 11 4 36.4% 7 KV (2doses) 12 5 41.7% 8 Challenge Controls 10 9 90.0% 9 Strict NegativeControls 11 10 90.9% vORF2 = isolated viral ORF2; rORF2 = recombinantbaculovirus expressed ORF2; KV or killed whole cell virus = PCV2 virusgrown in suitable cell culture

Post-challenge, Group 5, which received one dose of 8 μg of rORF2antigen, outperformed the other 6 vaccine groups. Group 5 had thehighest ADWG (0.94±0.22 lbs/day), the lowest incidence of abnormalbehavior (0%), the second lowest incidence of cough (8.3%), the lowestincidence of overall clinical symptoms (8.3%), the lowest mortality rate(0%), the lowest rate of nasal shedding of PCV2 (8.3%), the secondlowest rate for mean % lung lesions (0.68±1.15%) and the lowestincidence rate for positive tissues (16.7%). Groups receiving variouslevels of rORF2 antigen overall outperformed groups receiving variouslevels of vORF2 and the group receiving 2 doses of killed whole cellPCV2 vaccine performed the worst. Tables 14 and 15 contain summaries ofgroup post-challenge data.

TABLE 14 Summary of Group Post-Challenge Data—Part 1 Overall IncidenceADWG Abnormal of Clinical Group N Treatment (lbs/day) Behavior CoughSymptoms 1 12 vORF2— 0.87 ± 0.29 2/12 3/12 41.7% 16 μg (16.7%) (25%) (1dose) 2 12 vORF2— 0.70 ± 0.32 4/12 1/12 41.7% 8 μg (33.3%) (8.3%) (1dose) 3 12 vORF2— 0.49 ± 0.21 8/12 2/12 66.7% 4 μg (66.7%) (16.7%) (1dose) 4 11 rORF2— 0.84 ± 0.30 3/11 0/11 36.4% 16 μg (27.3%) (0%) (1dose) 5 12 rORF2— 0.94 ± 0.22 0/12 1/12  8.3% 8 μg (0%) (8.3%) (1 dose)6 11 rORF2— 0.72 ± 0.25 1/11 0/11  9.1% 4 μg (9.1%) (0%) (1 dose) 7 12KV 0.50 ± 0.15 7/12 0/12 58.3% (2 doses) (58.3) (0%) 8 10 Challenge 0.76± 0.19 1/10 2/10   40% Controls (10%) (20%) 9 11 Strict 1.06 ± 0.17 0/110/11   0% Negative (0%) (0%) Controls vORF2 = isolated viral ORF2; rORF2= recombinant baculovirus expressed ORF2; KV or killed whole cell virus= PCV2 virus grown in suitable cell culture

TABLE 15 Summary of Group Post-Challenge Data—Part 2 Incidence Rate ofat least one tissue Mean % IHC Mortality Nasal Lung positive Group NTreatment Rate Shedding Lesions for PCV2 1 12 vORF2—  8.3%  8.3% 0.40 ±0.50% 16.7% 16 μg (1 dose) 2 12 vORF2—  8.3%  8.3% 7.41 ± 20.2% 25.0% 8μg (1 dose) 3 12 vORF2—   0%  8.3% 9.20 ± 20.9% 66.7% 4 μg (1 dose) 4 11rORF2—   0% 18.2% 1.50 ± 4.74% 36.3% 16 μg (1 dose) 5 12 rORF2—   0% 8.3% 0.68 ± 1.15% 16.7% 8 μg (1 dose) 6 11 rORF2—  9.1%  9.1% 2.95 ±5.12% 36.4% 4 μg (1 dose) 7 12 KV 16.7% 41.7%  7.27 ± 22.9% 41.7% (2doses) 8 10 Challenge   10%   80%  9.88 ± 29.2% 90.0% Controls 9 11Strict   0% 63.6% 0/11 90.9% Negative (0%) Controls vORF2 = isolatedviral ORF2; rORF2 = recombinant baculovirus expressed ORF2; KV or killedwhole cell virus = PCV2 virus grown in suitable cell culture

Results of this study indicate that all further vaccine efforts shouldfocus on a rORF2 vaccine. Overall, nasal shedding of PCV2 was detectedpost-challenge and vaccination with a PCV2 vaccine resulted in areduction of shedding. Immunohistochemistry of selected lymphoid tissuesalso served as a good parameter for vaccine efficacy, whereas largedifferences in ADWG, clinical symptoms, and gross lesions were notdetected between groups. This study was complicated by the fact thatextraneous PCV2 was introduced at some point during the study, asevidenced by nasal shedding of PCV2, PCV2 seroconversion and positiveIHC tissues in Group 9, the strict negative control group.

Discussion

Seven PCV2 vaccines were evaluated in this study, which included threedifferent dose levels of vORF2 antigen administered once on Day 0, threedifferent dose levels of rORF2 antigen administered once on Day 0 andone dose level of killed whole cell PCV2 vaccine administered on Day 0and Day 14. Overall, Group 5, which received 1 dose of vaccinecontaining 8 μg of rORF2 antigen, had the best results. Group 5 had thehighest ADWG, the lowest incidence of abnormal behavior, the lowestincidence of abnormal respiration, the second lowest incidence of cough,the lowest incidence of overall clinical symptoms, the lowest mortalityrate, the lowest rate of nasal shedding of PCV2, the second lowest ratefor mean % lung lesions and the lowest incidence rate for positive IHCtissues.

Interestingly, Group 4, which received a higher dose of rORF2 antigenthan Group 5, did not perform as well or better than Group 5. Group 4had a slightly lower ADWG, a higher incidence of abnormal behavior, ahigher incidence of overall clinical symptoms, a higher rate of nasalshedding of PCV2, a higher mean % lung lesions, and a higher rate forpositive IHC tissues than Group 5. Statistical analysis, which may haveindicated that the differences between these two groups were notstatistically significant, was not conducted on these data, but therewas an observed trend that Group 4 did not perform as well as Group 5.

Post-vaccination, 6 pigs died at the first study site. Four of the sixpigs were from Group 8 or Group 9, which received no vaccine. None ofthe six pigs demonstrated lesions consistent with PMWS, no adverseevents were reported and overall, all seven vaccines appeared to be safewhen administered to pigs approximately 11 days of age. During thepost-vaccination phase of the study, pigs receiving either of three doselevels of vORF2 vaccine or killed whole cell vaccine had the highestIFAT levels, while Group 5 had the lowest IFAT levels just prior tochallenge, of the vaccine groups.

Although not formally proven, the predominant route of transmission ofPCV2 to young swine shortly after weaning is believed to be by oronasaldirect contact and an efficacious vaccine that reduces nasal shedding ofPCV2 in a production setting would help control the spread of infection.Groups receiving one of three vORF2 antigen levels and the groupreceiving 8 μg of rORF2 had the lowest incidence rate of nasal sheddingof PCV2 (8.3%). Expectedly, the challenge control group had the highestincidence rate of nasal shedding (80%).

Gross lesions in pigs with PMWS secondary to PCV2 infection typicallyconsist of generalized lymphadenopathy in combination with one or amultiple of the following: (1) interstitial pneumonia with interlobularedema, (2) cutaneous pallor or icterus, (3) mottled atrophic livers, (4)gastric ulcers and (5) nephritis. At necropsy, icterus, hepatitis,nephritis, and gastric ulcers were not noted in any groups andlymphadenopathy was not specifically examined for. The mean % lunglesion scores varied between groups. The group receiving 16 μg of vORF2antigen had the lowest mean % lung lesion score (0.40±0.50%), followedby the group that received 8 μg of rORF2 (0.68±1.15%). As expected, thechallenge control group had the highest mean % lung lesion score(9.88±29.2%). In all four groups, the mean % lung lesion scores wereelevated due to one pig in each of these groups that had very high lunglesion scores. Most of the lung lesions were described as red/purple andconsolidated. Typically, lung lesions associated with PMWS are describedas tan and non-collapsible with interlobular edema. The lung lesionsnoted in this study were either not associated with PCV2 infection or asecond pulmonary infectious agent may have been present. Within thecontext of this study, the % lung lesion scores probably do not reflecta true measure of the amount of lung infection due to PCV2.

Other researchers have demonstrated a direct correlation between thepresence of PCV2 antigen by IHC and histopathology. Histopathology onselect tissues was not conducted with this study. Group 1 (16 μg ofvORF2) and Group 5 (8 μg of rORF2) had the lowest incidence rate of pigspositive for PCV2 antigen (8.3%), while Group 9 (the strict negativecontrol group—90.9%) and Group 8 (the challenge control group—90.0%) hadthe highest incidence rates for pigs positive for PCV2 antigen. Due tothe non-subjective nature of this test, IHC results are probably one ofthe best parameters to judge vaccine efficacy on.

Thus, in one aspect of the present invention, the Minimum PortectiveDosage (MPD) of a 1 ml/l dose recombinant product with extracted PCV2ORF2 (rORF2) antigen in the CDCD pig model in the face of a PCV2challenge was determined Of the three groups that received varyinglevels of rORF2 antigen, Group 5 (8 μg of rORF2 antigen) clearly had thehighest level of protection. Group 5 either had the best results or wastied for the most favorable results with regard to all of the parametersexamined. When Group 5 was compared with the other six vaccine groupspost-challenge, Group 5 had the highest ADWG (0.94±0.22 lbs/day), thelowest incidence of abnormal behavior (0%), the second lowest incidenceof cough (8.3%), the lowest incidence of overall clinical symptoms(8.3%), the lowest mortality rate (0%), the lowest rate of nasalshedding of PCV2 (8.3%), the second lowest rate for mean % lung lesions(0.68±1.15%) and the lowest incidence rate for positive IHC tissues(16.7%).

In another aspect of the present invention, the MPD of a 1 ml/1 doseconventional product that is partially purified PCV2 ORF2 (vORF2)antigen in the CDCD pig model in the face of a PCV2 challenge wasdetermined Of the three groups that received varying levels of vORF2antigen, Group 1 (16 μg of vORF2) had the highest level of protection.Group 1 outperformed Groups 2 and 3 with respect to ADWG, mean % lunglesions, and IHC. Groups 1 and 2 (8 μg of vORF2 antigen) performedequally with respect to overall incidence of clinical symptoms, Group 3(4 μg of vORF2 antigen) had the lowest mortality rate, and all threegroups performed equally with respect to nasal shedding. Overall, vORFvaccines did not perform as well as rORF vaccines.

In yet another aspect of the present invention, the efficacy of amaximum dose of a 2 ml/2 dose Conventional Killed PCV2 vaccine in theCDCD pig model in the face of a PCV2 challenge was determined Of theseven vaccines evaluated in this study, the killed whole cell PCV2vaccine performed the worst. Piglets receiving two doses of killed wholecell PCV2 vaccine had the lowest ADWG, the second highest rate ofabnormal behavior (58.3%), the second highest overall incidence ofclinical symptoms (58.3%), the highest mortality rate (16.7%), thesecond highest incidence of nasal shedding (41.7%), highest mean % lunglesions (9.88±29.2%), a high incidence of lung lesions noted (75%) and amoderate IHC incidence rate in tissues (41.7%). However, it was stilleffective at invoking an immune response.

In still another aspect of the present invention, nasal shedding of PCV2was assessed as an efficacy parameter and the previous PCV2 efficacyparameters from previous studies were reconfirmed. Results from thisstudy indicate that nasal shedding of PCV2 occurs following intra nasalchallenge and that PCV2 vaccines reduce nasal shedding of PCV2post-challenge. Furthermore, results from this study and reports in theliterature indicate that IHC should continue to be evaluated in futurePCV2 vaccine trials as well.

Some additional conclusions arising from this study are thatlymphadenopathy is one of the hallmarks of PMWS. Another one of thehallmarks of PMWS is lymphoid depletion and multinucleated/gianthistiocytes. Additionally, no adverse events or injection site reactionswere noted for any of the 7 PCV2 vaccines and all 7 PCV2 vaccinesappeared to be safe when administered to young pigs.

Example 5

This example tests the efficacy of eight PCV2 candidate vaccines andreconfirms PCV2 challenge parameters from earlier challenge studiesfollowing exposure to a virulent strain of PCV2. One hundred and fifty(150) cesarean derived colostrum deprived (CDCD) piglets, 6-16 days ofage, were blocked by weight and randomly divided into 10 groups of equalsize. Table 16 sets forth the General Study Design for this Example.

TABLE 16 General Study Design KLH/ Challenge Day ICFA with PRRSV Nec-No. of on Day Virulent MLV ropsy Of Treat- 22 and PCV2 on on on GroupPigs Treatment ment Day 28 Day 25 Day 46 Day 50 1 15 PVC2 0 & 14 + + + +Vaccine 1 16 μg rORF2— IMS 1314 2 15 PVC2 0 & 14 + + + + Vaccine 2 16 μgvORF2— Carbopol 3 15 PCV2 0 & 14 + + + + Vaccine 3 16 μg rORF2— Carbopol4 15 PCV2 0 + + + + Vaccine 2 16 μg vORF2— Carbopol 5 15 PVC2 0 &14 + + + + Vaccine 3 4 μg rORF2— Carbopol 6 15 PVC2 0 & 14 + + + +Vaccine 3 1 μg rORF2— Carbopol 7 15 PVC2 0 & 14 + + + + Vaccine 3 0.25μg rORF2— Carbopol 8 15 PVC2 0 & 14 + + + + Vaccine 4 >8.0 log KV—Carbopol 9 15 Challenge N/A + + + + Controls 10 15 None— N/A + − + +Strict Negative Control Group vORF2 = isolated viral ORF2; rORF2 =recombinant baculovirus expressed ORF2; KV or killed whole cell virus =PCV2 virus grown in suitable cell culture

The vaccine formulation given to each group was as follows. PCV2 VaccineNo. 1, administered at 1×2 ml dose to Group 1, was a high dose (16 ug/2ml dose) of inactivated recombinant ORF2 antigen adjuvanted with IMS1314 (16 ug rORF2—IMS 1314). PCV2 Vaccine No. 2, administered at 1×2 mldose to Group 2, was a high dose (16 ug/2 ml dose) of a partiallypurified VIDO R-1 generated PCV2 ORF2 antigen adjuvanted with Carbopol(16 ug vORF2—Carbopol). PCV2 Vaccine No. 3, administered at 1×2 ml doseto Group 3, was a high dose (16 ug/2 ml dose) of inactivated recombinantORF2 antigen adjuvanted with Carbopol (16 ug rORF2—Carbopol). PCV2Vaccine No. 4, administered at 1×1 ml dose to Group 4, was a high dose(16 ug/1 ml dose) of a partially purified VIDO R-1 generated PCV2 ORF2antigen adjuvanted with Carbopol (16 ug vORF2—Carbopol). Vaccine No. 5,administered at 1×2 ml dose to Group 5, was a 4 ug/2 ml dose of aninactivated recombinant ORF2 antigen adjuvanted with Carbopol (4 ugrORF2—Carbopol). PCV2 Vaccine No. 6, administered at 1×2 ml dose toGroup 6, was a 1 ug/2 ml dose of an inactivated recombinant ORF2 antigenadjuvanted with Carbopol (1 ug rORF2—Carbopol). PCV2 Vaccine No. 7,administered at 1×2 ml dose to Group 7, was a low dose (0.25 ug/2 mldose) of inactivated recombinant ORF2 antigen adjuvanted with Carbopol(0.25 ug rORF2—Carbopol). PCV2 Vaccine No. 8, administered at 1×2 mldose to Group 8, was a high dose (pre-inactivation titer >8.0 log/2 mldose) Inactivated Conventional Killed VIDO R-1 generated PCV2 Struveantigen adjuvanted with Carbopol (>8.0 log KV—Carbopol). On Day 0,Groups 1-8 were treated with their assigned vaccines. Groups 1-3 and 5-8received boosters of their respective vaccines again on Day 14. Theeffectiveness of a single dose of 16 ng of vORF2—Carbopol was tested onGroup 4 which did not receive a booster on Day 14. Piglets were observedfor adverse events and injection site reactions following bothvaccinations. On Day 21 the piglets were moved to a second study sitewhere Groups 1-9 were group housed in one building and Group 10 washoused in a separate building. All pigs received keyhole limpethemocyanin emulsified with incomplete Freund's adjuvant (KLH/ICFA) onDays 22 and 28. On Day 25, Groups 1-9 were challenged with approximately4 logs of virulent PCV2 virus. By Day 46, very few deaths had occurredin the challenge control group. In an attempt to immunostimulate thepigs and increase the virulence of the PCV2 challenge material, allGroups were treated with INGELVAC® PRRSV MLV (Porcine Reproductive andRespiratory Vaccine, Modified Live Virus) on Day 46.

Pre- and post-challenge blood samples were collected for PCV2 serology.Post-challenge, body weight data for determination of average dailyweight gain (ADWG) and observations of clinical signs were collected. OnDay 50, all surviving pigs were necropsied, gross lesions were recorded,lungs were scored for pathology, and selected tissues were preserved informalin for examination by Immunohistochemistry (IHC) for detection ofPCV2 antigen at a later date.

Materials and Methods

This was a partially-blind vaccination-challenge feasibility studyconducted in CDCD pigs, 6 to 16 days of age on Day 0. To be included inthe study, PCV2 IFA titers of sows were <1:1000. Additionally, theserologic status of sows were from a known PRRS-negative herd. Sixteen(16) sows were tested for PCV2 serological status and all sixteen (16)had a PCV2 titer of <1000 and were transferred to the first study site.One hundred fifty (150) piglets were delivered by cesarean sectionsurgeries and were available for this study on Day −3. On Day −3, 150CDCD pigs at the first study site were weighed, identified with eartags, blocked by weight and randomly assigned to 1 of 10 groups, as setforth above in table 16. Blood samples were collected from all pigs. Ifany test animal meeting the inclusion criteria was enrolled in the studyand was later excluded for any reason, the Investigator and Monitorconsulted in order to determine the use of data collected from theanimal in the final analysis. The date of which enrolled piglets wereexcluded and the reason for exclusion was documented. No sows meetingthe inclusion criteria, selected for the study and transported to thefirst study site were excluded. No piglets were excluded from the study,and no test animals were removed from the study prior to termination.Table 17 describes the time frames for the key activities of thisExample.

TABLE 17 Study Activities Study Day Actual Dates Study Activity −34-04-03 Weighed pigs; health exam; randomized to groups; collected bloodsamples −3, 4-04-03 Observed for overall health and for adverse 0-214-07-03 to events post-vaccination 5-27-03 0 4-07-03 Administeredrespective IVPs to Groups 1-8 0-7 4-07-03 to Observed pigs for injectionsite reactions 4-14-03 14 4-21-03 Boostered Groups 1-3, 5-8 withrespective IVPs; blood sampled all pigs 14-21 4-21-03 to Observed pigsfor injection reactions 4-28-03 19-21 4-26-03 to Treated all pigs withantibiotics 4-28-03 21 4-28-03 Pigs transported from Struve Labs, Inc.to Veterinary Resources, Inc.(VRI) 22-50 4-28-03 to Observed pigs forclinical signs post-challenge 5-27-03 22 4-29-03 Treated Groups 1-10with KLH/ICFA 25 5-02-03 Collected blood samples from all pigs; weighedall pigs; challenged Groups 1-9 with PCV2 challenge material 28 5-05-03Treated Groups 1-10 with KLH/ICFA 32 5-09-03 Collected blood samplesfrom all pigs 46 5-23-03 Administered INGELVAC ® PRRS MLV to all groups50 5-27-03 Collected blood samples, weighed and necropsied all pigs;gross lesions were recorded; lungs were evaluated for lesions; fresh andformalin fixed tissue samples were saved; In-life phase of the study wascompleted

Following completion of the in-life phase of the study, formalin fixedtissues were examined by Immunohistochemistry (IHC) for detection ofPCV2 antigen by a pathologist, blood samples were evaluated for PCV2serology, and average daily weight gain (ADWG) was determined from Day25 to Day 50.

Animals were housed at the first study site in individual cages in sevenrooms from birth to approximately 11 days of age (approximately Day 0 ofthe study). Each room was identical in layout and consisted of stackedindividual stainless steel cages with heated and filtered air suppliedseparately to each isolation unit. Each room had separate heat andventilation, thereby preventing cross-contamination of air betweenrooms. Animals were housed in two different buildings at the secondstudy site. Group 10 (The Strict negative control group) was housedseparately in a converted nursery building and Groups 1-9 were housed ina converted farrowing building. Each group was housed in a separate pen(14-15 pigs per pen) and each pen provided approximately 2.3 square feetper pig. Groups 2, 4 and 8 were penned in three adjacent pens on oneside of the alleyway and Groups 1, 3, 5, 6, 7, and 9 were penned in sixadjacent pens on the other side of the alleyway. The Group separationwas due to concern by the Study Monitor that vaccines administered toGroups 2, 4, and 8 had not been fully inactivated. Each pen was on anelevated deck with plastic slatted floors. A pit below the pens servedas a holding tank for excrement and waste. Each building had its ownseparate heating and ventilation systems, with little likelihood ofcross-contamination of air between buildings.

At the first study site, piglets were fed a specially formulated milkration from birth to approximately 3 weeks of age. All piglets wereconsuming solid, special mixed ration by Day 21 (approximately 4½ weeksof age). At the second study site, all piglets were fed a customnon-medicated commercial mix ration appropriate for their age andweight, ad libitum. Water at both study sites was also available adlibitum.

All test pigs were treated with 1.0 mL of NAXCEL®, IM, in alternatinghams on Days 19, 20, and 21. In addition, Pig No. 11 (Group 1) wastreated with 0.5 mL of NAXCEL® IM on Day 10, Pig No. 13 (Group 10) wastreated with 1 mL of Penicillin and 1 mL of PREDEF® 2× on Day 10, PigNo. 4 (Group 9) was treated with 1.0 mL of NAXCEL® IM on Day 11, andPigs 1 (Group 1), 4 and 11 were each treated with 1.0 mL of NAXCEL® onDay 14 for various health reasons.

While at both study sites, pigs were under veterinary care. Animalhealth examinations were conducted on Day −3 and were recorded on theHealth Examination Record Form. All animals were in good health andnutritional status before vaccination as determined by observation onDay 0. All test animals were observed to be in good health andnutritional status prior to challenge. Carcasses and tissues weredisposed of by rendering. Final disposition of study animals wasrecorded on the Animal Disposition Record.

On Days 0 and 14, pigs assigned to Groups 1-3 and 5-8 received 2.0 mL ofassigned PCV2 Vaccines 1-4, respectively, IM in the right and left neckregion, respectively, using a sterile 3.0 mL Luer-lock syringe and asterile 20 g×½″ needle. Pigs assigned to Group 4 received 1.0 mL of PCV2Vaccine No. 2, IM in the right neck region using a sterile 3.0 mLLuer-lock syringe and a sterile 20 g×½″ needle on Day 0 only.

On Day 22 all test pigs received 2.0 mL of KLH/ICFA IM in the left neckregion using a sterile 3.0 mL Luer-lock syringe and a sterile 20 g×1″needle. On Day 28 all test pigs received 2.0 mL of KLH/ICFA in the rightham region using a sterile 3.0 mL Luer-lock syringe and a sterile 20g×1″ needle.

On Day 25, pigs assigned to Groups 1-9 received 1.0 mL of PCV2 ISUVDLchallenge material (3.98 log₁₀ TCID₅₀/mL) IM in the right neck regionusing a sterile 3.0 mL Luer-lock syringe and a sterile 20 g×1″ needle.An additional 1.0 mL of the same material was administered IN to eachpig (0.5 mL per nostril) using a sterile 3.0 mL Luer-lock syringe andnasal canula.

On Day 46, all test pigs received 2.0 mL INGELVAC® PRRS MLV, IM, in theright neck region using a sterile 3.0 mL Luer0lock syringe and a sterile20 g×1″ needle. The PRRSV MLV was administered in an attempt to increasevirulence of the PCV2 challenge material.

Test pigs were observed daily for overall health and adverse events onDay −3 and from Day 0 to Day 21. Each of the pigs was scored for normalor abnormal behavior, respiration or cough. Observations were recordedon the Clinical Observation Record. All test pigs were observed from Day0 to Day 7, and Group 7 was further observed from Day 14 to 21, forinjection site reactions. Average daily weight gain was determined byweighing each pig on a calibrated scale on Days −3, 25 and 50, or on theday that a pig was found dead after challenge. Body weights wererecorded on the Body Weight Form. Day −3 body weights were utilized toblock pigs prior to randomization. Day 25 and Day 50 weight data wasutilized to determine the average daily weight gain (ADWG) for each pigduring these time points. For pigs that died after challenge and beforeDay 50, the ADWG was adjusted to represent the ADWG from Day 25 to theday of death.

In order to determine PCV2 serology, venous whole blood was collectedfrom each piglet from the orbital venous sinus on Days −3 and 14. Foreach piglet, blood was collected from the orbital venous sinus byinserting a sterile capillary tube into the medial canthus of one of theeyes and draining approximately 3.0 mL of whole blood into a 4.0 mLSerum Separator Tube (SST). On Days 25, 32, and 50, venous whole bloodfrom each pig was collected from the anterior vena cava using a sterile20 g×1½″ Vacutainer® needle (Becton Dickinson and Company, FranklinLakes, N.J.), a Vaccutainer® needle holder and a 13 mL SST. Bloodcollections at each time point were recorded on the Sample CollectionRecord. Blood in each SST was allowed to clot, each SST was then spundown and the serum harvested. Harvested serum was transferred to asterile snap tube and stored at −70±10° C. until tested at a later date.Serum samples were tested for the presence of PCV2 antibodies byBIVI-R&D personnel.

Pigs were observed once daily from Day 22 to Day 50 for clinicalsymptoms and scored for normal or abnormal behavior, respiration orcough. Clinical observations were recorded on the Clinical ObservationRecord.

Pigs Nos. 46 (Group 1) and 98 (Groups 9) died at the first study site.Both of these deaths were categorized as bleeding deaths and necropsieswere not conducted on these two pigs. At the second study site, pigsthat died after challenge and prior to Day 50, and pigs euthanized onDay 50, were necropsied. Any gross lesions were noted and thepercentages of lung lobes with lesions were recorded on the NecropsyReport Form.

From each of the pigs necropsied at the second study site, a tissuesample of tonsil, lung, heart, and mesenteric lymph node was placed intoa single container with buffered 10% formalin; while another tissuesample from the same aforementioned organs was placed into a Whirl-Pak®(M-Tech Diagnostics Ltd., Thelwall, UK) and each Whirl-Pak® was placedon ice. Each container was properly labeled. Sample collections wererecorded on the Necropsy Report Form. Afterwards, formalin-fixed tissuesamples and a Diagnostic Request Form were submitted for IHC testing.IHC testing was conducted in accordance with standard laboratoryprocedures for receiving samples, sample and slide preparation, andstaining techniques. Fresh tissues in Whirl-Paks® were shipped with icepacks to the Study Monitor for storage (−70°±10° C.) and possible futureuse.

Formalin-fixed tissues were examined by a pathologist for detection ofPCV2 by IHC and scored using the following scoring system: 0=None;1=Scant positive staining, few sites; 2=Moderate positive staining,multiple sites; and 3=Abundant positive staining, diffuse throughout thetissue. For analytical purposes, a score of 0 was considered “negative,”and a score of greater than 0 was considered “positive.”

Results

Results for this example are given below. It is noted that Pigs No. 46and 98 died on days 14 and 25 respectively. These deaths werecategorized as bleeding deaths. Pig No. 11 (Group 1) was panting withrapid respiration on Day 15. Otherwise, all pigs were normal forbehavior, respiration and cough during this observation period and nosystemic adverse events were noted with any groups. No injection sitereactions were noted following vaccination on Day 0. Followingvaccination on Day 14, seven (7) out of fourteen (14) Group 1 pigs(50.0%) had swelling with a score of “2” on Day 15. Four (4) out offourteen (14) Group 1 (28.6%) still had a swelling of “2” on Day 16.None of the other groups experienced injection site reactions followingeither vaccination.

Average daily weight gain (ADWG) results are presented below in Table18. Pigs No. 46 and 98 that died from bleeding were excluded from groupresults. Group 4, which received one dose of 16 ug vORF2—Carbopol, hadthe highest ADWG (1.16±0.26 lbs/day), followed by Groups 1, 2, 3, 5, 6,and 10 which had ADWGs that ranged from 1.07±0.23 lbs/day to 1.11±0.26lbs/day. Group 9 had the lowest ADWG (0.88±0.29 lbs/day), followed byGroups 8 and 7, which had ADWGs of 0.93±0.33 lbs/day and 0.99±0.44lbs/day, respectively.

TABLE 18 Summary of Group Average Daily Weight Gains (ADWG) ADWG-lbs/day(Day 25 to Day 50) or adjusted for pigs Group Treatment N dead beforeDay 50 1 rORF2-16 μg-IMS 1314 2 doses 14 1.08 ± 0.30 lbs/day 2 vORF2-16μg-Carbopol 2 doses 15 1.11 ± 0.16 lbs/day 3 rORF2-16 μg-Carbopol 2doses 15 1.07 ± 0.21 lbs/day 4 vORF2-16 μg-Carbopol 1 dose 15 1.16 ±0.26 lbs/day 5 rORF2-4 μg-Carbopol 1 dose 15 1.07 ± 0.26 lbs/day 6rORF2-1 μg-Carbopol 2 doses 15 1.11 ± 0.26 lbs/day 7 rORF2-0.25μg-Carbopol 2 doses 15 0.99 ± 0.44 lbs/day 8 KV > 8.0 log-Carbopol 2doses 15 0.93 ± 0.33 lbs/day 9 Challenge Controls 14 0.88 ± 0.29 lbs/day10 Strict Negative Controls 15 1.07 ± 0.23 lbs/day vORF2 = isolatedviral ORF2; rORF2 = recombinant baculovirus expressed ORF2; KV or killedwhole cell virus = PCV2 virus grown in suitable cell culture

PVC2 serology results are presented below in Table 19. All ten (10)groups were seronegative for PCV2 on Day −3. On Day 14, PCV2 titersremained low for all ten (10) groups (range of 50-113). On Day 25, Group8, which received the whole cell killed virus vaccine, had the highestPCV2 titer (4617), followed by Group 2, which received 16 ugvORF2—Carbopol, Group 4, which received as single dose of 16 ugvORF2—Carbopol, and Group 3, which received 16 ug rORF2—Carbopol, whichhad titers of 2507, 1920 and 1503 respectively. On Day 32 (one week postchallenge), titers for Groups 1-6 and Group 8 ranged from 2360 to 7619;while Groups 7 (0.25 ug rORF2—Carbopol), 9 (Challenge Control), and 10(Strict negative control) had titers of 382, 129 and 78 respectively. OnDay 50 (day of necropsy), all ten (10) groups demonstrated high PCV2titers (>1257).

On Days 25, 32, and 50, Group 3, which received two doses of 16 ugrORF2—Carbopol had higher antibody titers than Group 1, which receivedtwo doses of 16 ug rORF2—IMS 1314. On Days 25, 32 and 50, Group 2, whichreceived two doses of 16 ug vORF2 had higher titers than Group 4, whichreceived only one does of the same vaccine. Groups 3, 5, 6, 7, whichreceived decreasing levels of rORF2—Carbopol, of 16, 4, 1, and 0.25 ugrespectively, demonstrated correspondingly decreasing antibody titers onDays 25 and 32.

TABLE 19 Summary of Group PCV2 IFA Titers Day Day Day Day Day GroupTreatment −3 14** 25*** 32 50**** 1 rORF2—16 μg— 50 64 646 3326 4314 IMS1314 2 doses 2 vORF2—16 μg— 50 110 2507 5627 4005 Carbopol 2 doses 3rORF2—16 μg— 50 80 1503 5120 6720 Carbopol 2 doses 4 vORF2—16 μg— 50 1131920 3720 1257 Carbopol 1 dose 5 rORF2—4 μg— 50 61 1867 3933 4533Carbopol 1 dose 6 rORF2—1 μg— 50 70 490 2360 5740 Carbopol 2 doses 7rORF2—0.25 μg 50 73 63 382 5819 Carbopol 2 doses 8 KV > 8.0 log— 50 974617 7619 10817 Carbopol 2 doses 9 Challenge Controls 50 53 50 129 428810 Strict Negative Controls 50 50 50 78 11205 vORF2 = isolated viralORF2; rORF2 = recombinant baculovirus expressed ORF2; KV or killed wholecell virus = PCV2 virus grown in suitable cell culture *For calculationpurposes, a ≦100 IFA titer was designated as a titer of “50”; a ≧6400IFA titer was designated as a titer of “12,800”. **Day of Challenge***Day of Necropsy

The results from the post-challenge clinical observations are presentedbelow. Table 20 includes observations for Abnormal Behavior, AbnormalRespiration, Cough and Diarrhea. Table 21 includes the results from theSummary of Group Overall Incidence of Clinical Symptoms and Table 22includes results from the Summary of Group Mortality RatesPost-challenge. The incidence of abnormal behavior, respiration andcough post-challenge were low in pigs receiving 16 ug rORF2—IMS 1314(Group 1), 16 ug rORF2—Carbopol (Group 3), 1 ug rORF2—Carbopol (Group6), 0.25 ug rORF2—Carbopol (Group 7), and in pigs in the ChallengeControl Group (Group 9). The incidence of abnormal behavior respirationand cough post-challenge was zero in pigs receiving 16 ug vORF2—Carbopol(Group 2), a single dose of 16 ug vORF2—Carbopol (Group 4), 4 ugrORF2—Carbopol (Group 5), >8 log KV-Carbopol (Group 8), and in pigs inthe strict negative control group (Group 10).

The overall incidence of clinical symptoms varied between groups. Pigsreceiving 16 ug vORF2—Carbopol (Group 2), a single dose of 16 ugvORF2—Carbopol (Group 4), and pigs in the Strict negative control group(Group 10) had incidence rates of 0%; pigs receiving 16 ugrORF2—Carbopol (Group 3), and 1 ug rORF2—Carbopol (Group 6) hadincidence rates of 6.7%; pigs receiving 16 ug rORF2—IMS 1314 (Group 1)had an overall incidence rate of 7.1%; pigs receiving 4 ugrORF2—Carbopol (Group 5), 0.25 ug rORF2—Carbopol (Group 7), and >8 logKV vaccine had incidence rates of 13.3%; and pigs in the ChallengeControl Group (Group 9) had an incidence rate of 14.3%.

Overall mortality rates between groups varied as well. Group 8, whichreceived 2 doses of KV vaccine had the highest mortality rate of 20.0%;followed by Group 9, the challenge control group, and Group 7, whichreceived 0.25 ug rORF2—Carbopol and had mortality rates of 14.3% and13.3% respectively. Group 4, which received one dose of 16 ugvORF2—Carbopol had a 6.7% mortality rate. All of the other Groups, 1, 2,3, 5, 6, and 10 had a 0% mortality rate.

TABLE 20 Summary of Group Observations for Abnormal Behavior, AbnormalRespiration, and Cough Post-Challenge Abnormal Abnormal Group TreatmentN Behavior¹ Behavior² Cough³ 1 rORF2-16 μg- 14 0/14 0/14 1/14 IMS 1314 2doses (0%) (0%) (7.1%) 2 vORF2-16 μg-Carbopol 15 0/15 0/15 0/15 2 doses( 0%) (0%) (0%) 3 rORF2-16 μg-Carbopol 15 0/15 0/15 1/15 2 doses (0%)(0%) (6.7%) 4 vORF2-16 μg-Carbopol 15 0/15 0/15 0/15 1 dose (0%) (0%)(0%) 5 rORF2-4 μg- 15 1/15 1/15 0/15 Carbopol 1 dose (6.7%) (6.7%) (0%)6 rORF2-1 μg- 15 0/15 0/15 1/15 Carbopol 2 doses (0%) (0%) (6.7%) 7rORF2-0.25 μg- 15 0/15 1/15 1/15 Carbopol 2 doses (0%) (6.7%) (06.7%) 8KV > 8.0 log-Carbopol 2 15 1/15 1/15 0/15 doses (6.7%) (6.7%) (0%) 9Challenge Controls 14 1/14 1/14 2/14 (7.1%) (7.1%) (14/3%) 10 StrictNegative Controls 15 0/15 0/15 0/15 (0%) (0%) (0%) ¹Total number of pigsin each group that demonstrated any abnormal behavior for at least oneday ²Total number of pigs in each group that demonstrated any abnormalrespiration for at least one day ³Total number of pigs in each groupthat demonstrated a cough for at least one day

TABLE 21 Summary of Group Overall Incidence of Clinical Symptoms Post-Challenge Incidence of pigs with Incidence Group Treatment N ClinicalSymptoms¹ Rate 1 rORF2-16 μg- 14 1  7.1% IMS 1314 2 doses 2 vORF2-16μg-Carbopol 2 15 0  0.0% doses 3 rORF2-16 μg-Carbopol 2 15 1  6.7% doses4 vORF2-16 μg-Carbopol 1 15 0  0.0% dose 5 rORF2-4 μg- 15 2 13.3%Carbopol 1 dose 6 rORF2-1 μg- 15 1  6.7% Carbopol 2 doses 7 rORF2-0.25μg-Carbopol 15 2 13.3% 2 doses 8 KV > 8.0 log-Carbopol 2 15 2 13.3%doses 9 Challenge Controls 14 2 14.3% 10 Strict Negative Controls 15 0 0.0% vORF2 = isolated viral ORF2; rORF2 = recombinant baculovirusexpressed ORF2; KV or killed whole cell virus = PCV2 virus grown insuitable cell culture ¹Total number of pigs in each group thatdemonstrated any clinical symptom for at least one day¹ Total number of pigs in each group that demonstrated any clinicalsymptom for at least one day

TABLE 22 Summary of Group Mortality Rates Post-Challenge Dead Post-Mortality Group Treatment N challenge Rate 1 rORF2-16 μg- 14 0  0.0% IMS1314 2 doses 2 vORF2-16 μg-Carbopol 2 15 0  0.0% doses 3 rORF2-16μg-Carbopol 2 15 0  0.0% doses 4 vORF2-16 μg-Carbopol 1 15 1  6.7% dose5 rORF2-4 μg- 15 0  0.0% Carbopol 1 dose 6 rORF2-1 μg- 15 0  0.0%Carbopol 2 doses 7 rORF2-0.25 μg-Carbopol 2 15 2 13.3% doses 8 KV > 8.0log-Carbopol 2 15 3 20.0% doses 9 Challenge Controls 14 2 14.3% 10Strict Negative Controls 15 0  0.0% vORF2 = isolated viral ORF2; rORF2 =recombinant baculovirus expressed ORF2; KV or killed whole cell virus =PCV2 virus grown in suitable cell culture

The Summary of Group Mean Percentage Lung Lesions and TentativeDiagnosis is given below in Table 23. Group 9, the challenge controlgroup, had the highest percentage lung lesions with a mean of10.81±23.27%, followed by Group 7, which received 0.25 ug rORF2—Carbopoland had a mean of 6.57±24.74%, Group 5, which received 4 ugrORF2—Carbopol and had a mean of 2.88±8.88%, and Group 8, which receivedthe KV vaccine and had a mean of 2.01±4.98%. The remaining six (6)groups had lower mean percentage lung lesions that ranged from0.11±0.38% to 0.90±0.15%.

Tentative diagnosis of pneumonia varied among the groups. Group 3, whichreceived two doses of 16 ug rORF2—Carbopol, had the lowest tentativediagnosis of pneumonia, with 13.3%. Group 9, the challenge controlgroup, had 50% of the group tentatively diagnosed with pneumonia,followed by Group 10, the strict negative control group and Group 2,which received two doses of 16 ug vORF2—Carbopol, with 46.7% of 40%respectively, tentatively diagnosed with pneumonia.

Groups 1, 2, 3, 5, 9, and 10 had 0% of the group tentatively diagnosedas PCV2 infected; while Group 8, which received two doses if KV vaccine,had the highest group rate of tentative diagnosis of PCV2 infection,which 20%. Group 7, which received two doses of 0.25 ug rORF2—Carbopol,and Group 4, which received one dose of 16 ug vORF2—Carbopol hadtentative group diagnoses of PCV2 infection in 13.3% and 6.7% of eachgroup, respectively.

Gastric ulcers were only diagnosed in one pig in Group 7 (6.7%); whilethe other 9 groups remained free of gastric ulcers.

TABLE 23 Summary of Group Mean % Lung Lesion and Tentative Diagnosis No.Of pigs that shed for Incidence Group Treatment N at least one day Rate1 rORF2-16 μg- 15 0    0% IMS 1314 2 doses 2 vORF2-16 μg- 15 1  6.7%Carbopol 2 doses 3 rORF2-16 μg- 15 3  20.0% Carbopol 2 doses   4vORF2-16 μg- 15 2  13.3% Carbopol 1 dose   5 rORF2-4 μg- 15 3  20.0%Carbopol 1 dose   6 rORF2-1 μg- 15 6  40.0% Carbopol 2 doses   7rORF2-0.25 μg- 15 7  46.7% Carbopol 2 doses 8 KV > 8.0 log-Carbopol 1512   80% 2 doses 9 Challenge Controls 14 14 100.0% 10 Strict NegativeControls 15 14  93.3% vORF2 = isolated viral ORF2; rORF2 = recombinantbaculovirus expressed ORF2; KV or killed whole cell virus = PCV2 virusgrown in suitable cell culture

The Summary of Group IHC Positive Incidence Results are shown below inTable 24. Group 1 (16 ug rORF2—IMS 1314) had the lowest group rate ofIHC positive results with 0% of the pigs positive for PCV2, followed byGroup 2 (16 ug vORF2—Carbopol) and Group 4 (single dose 16 ugvORF2—Carbopol), which had group IHC rates of 6.7% and 13.3%respectively. Group 9, the challenge control group, had the highest IHCpositive incidence rate with 100% of the pigs positive for PCV2,followed by Group 10, the strict negative control group, and Group 8 (KVvaccine), with 93.3% and 80% of the pigs positive for PCV2,respectively.

Table 24. Summary of Group IHC Positive Incidence Rate

TABLE 24 Summary of Group IHC Positive Incidence Rate No. Of pigs thatshed for Incidence Group Treatment N at least one day Rate 1 rORF2-16μg- 15 0    0% IMS 1314 2 doses 2 vORF2-16 μg- 15 1  6.7% Carbopol 2doses 3 rORF2-16 μg- 15 3  20.0% Carbopol 2 doses   4 vORF2-16 μg- 15 2 13.3% Carbopol 1 dose   5 rORF2- 4 μg- 15 3  20.0% Carbopol 1 dose   6rORF2- 1 μg- 15 6  40.0% Carbopol 2 doses   7 rORF2-0.25 μg- 15 7  46.7%Carbopol 2 doses 8 KV > 8.0 log-Carbopol 15 12   80% 2 doses 9 ChallengeControls 14 14 100.0% 10 Strict Negative Controls 15 14  93.3% vORF2 =isolated viral ORF2; rORF2 = recombinant baculovirus expressed ORF2; KVor killed whole cell virus = PCV2 virus grown in suitable cell culture

Discussion

Seven PCV2 vaccines were evaluated in this example, which included ahigh dose (16 Kg) of rORF2 antigen adjuvanted with IMS 1314 administeredtwice, a high dose (16 μg) of vORF2 antigen adjuvanted with Carbopoladministered once to one group of pigs and twice to a second group ofpigs, a high dose (16 μg) of rORF2 antigen adjuvanted with Carbopoladministered twice, a 4 μg dose of rORF2 antigen adjuvanted withCarbopol administered twice, a 1 μg dose of rORF2 antigen adjuvantedwith Carbopol administered twice, a low dose (0.25 μg) of rORF2 antigenadjuvanted with Carbopol administered twice, and a high dose (>8 log) ofkilled whole cell PCV2 vaccine adjuvanted with Carbopol. Overall, Group1, which received two doses of 16 μg rORF2—IMS 1314, performed slightlybetter than Groups 2 through 7, which received vaccines containingvarious levels of either vORF2 or rORF2 antigen adjuvanted with Carbopoland much better than Group 8, which received two doses of killed wholecell PCV2 vaccine. Group 1 had the third highest ADWG (1.80±0.30lbs/day), the lowest incidence of abnormal behavior (0%), the lowestincidence of abnormal respiration (0%), a low incidence of cough (7.1%),a low incidence of overall clinical symptoms (7.1%), was tied with threeother groups for the lowest mortality rate (0%), the second lowest ratefor mean % lung lesions (0.15±0.34%), the second lowest rate forpneumonia (21.4%) and the lowest incidence rate for positive IHC tissues(0%). Group 1 was, however, the only group in which injection sitereactions were noted, which included 50% of the vaccinates 1 day afterthe second vaccination. The other vaccines administered to Groups 2through 7 performed better than the killed vaccine and nearly as well asthe vaccine administered to Group 1.

Group 8, which received two doses of killed PCV2 vaccine adjuvanted withCarbopol, had the worst set of results for any vaccine group. Group 8had the lowest ADWG (0.93±0.33 lbs/day), the second highest rate ofabnormal behavior (6.7%), the highest rate of abnormal respiration(6.7%), was tied with three other groups for the highest overallincidence rate of clinical symptoms (13.3%), had the highest mortalityrate of all groups (20%), and had the highest positive IHC rate (80%) ofany vaccine group. There was concern that the killed whole cell PCV2vaccine may not have been fully inactivated prior to administration toGroup 8, which may explain this group's poor results. Unfortunately,definitive data was not available to confirm this concern. Overall, inthe context of this example, a Conventional Killed PCV2 vaccine did notaid in the reduction of PCV2 associated disease.

As previously mentioned, no adverse events were associated with the testvaccines with exception of the vaccine adjuvanted with IMS 1314.Injection site reactions were noted in 50.0% of the pigs 1 day after thesecond vaccination with the vaccine formulated with IMS 1314 and in28.6% of the pigs 2 days after the second vaccination. No reactions werenoted in any pigs receiving Carbopol adjuvanted vaccines. Any furtherstudies that include pigs vaccinated with IMS 1314 adjuvanted vaccinesshould continue to closely monitor pigs for injection site reactions.

All pigs were sero-negative for PCV2 on Day −3 and only Group 2 had atiter above 100 on Day 14. On Day 25 (day of challenge), Group 8 had thehighest PCV2 antibody titer (4619), followed by Group 2 (2507). With theexception of Groups 7, 9 and 10, all groups demonstrated a strongantibody response by Day 32. By Day 50, all groups including Groups 7, 9and 10 demonstrated a strong antibody response.

One of the hallmarks of late stage PCV2 infection and subsequent PMWSdevelopment is growth retardation in weaned pigs, and in severe cases,weight loss is noted. Average daily weight gain of groups is aquantitative method of demonstrating growth retardation or weight loss.In this example, there was not a large difference in ADWG betweengroups. Group 8 had the lowest ADWG of 0.88±0.29 lbs/day, while Group 4had the highest ADWG of 1.16±0.26 lb/day. Within the context of thisstudy there was not a sufficient difference between groups to basefuture vaccine efficacy on ADWG.

In addition to weight loss—dyspnea, lethargy, pallor of the skin andsometimes icterus are clinical symptoms associated with PMWS. In thisexample, abnormal behavior and abnormal respiration and cough were notedinfrequently for each group. As evidenced in this study, this challengemodel and challenge strain do not result in overwhelming clinicalsymptoms and this is not a strong parameter on which to base vaccineefficacy.

Overall, mortality rates were not high in this example and the lack of ahigh mortality rate in the challenge control group limits this parameteron which to base vaccine efficacy. Prior to Day 46, Groups 4 and 7 eachhad one out of fifteen pigs die, Group 9 had two out of fourteen pigsdie and Group 8 had three out of fifteen pigs die. Due to the fact thatGroup 9, the challenge control group was not demonstrating PCV2 clinicalsymptoms and only two deaths had occurred in this group by Day 46,Porcine Respiratory and Reproductive Syndrome Virus (PRRSV) MLV vaccinewas administered to all pigs on Day 46. Earlier studies had utilizedINGELVAC® PRRS MLV as an immunostimulant to exasperate PCV2-associatedPMWS disease and mortality rates were higher in these earlier studies.Two deaths occurred shortly after administering the PRRS vaccine on Day46—Group 4 had one death on Day 46 and Group 7 had one death on Day47—which were probably not associated with the administration of thePRRS vaccine. By Day 50, Group 8, which received two doses of killedvaccine, had the highest mortality rate (20%), followed by Group 9(challenge control) and Group 7 (0.25 ug rORF2—Carbopol), with mortalityrates of 14.3% and 13.3% respectively. Overall, administration of thePRRS vaccine to the challenge model late in the post-challengeobservation phase of this example did not significantly increasemortality rates.

Gross lesions in pigs with PMWS secondary to PCV2 infection typicallyconsist of generalized lymphadenopathy in combination with one or moreof the following: (1) interstitial pneumonia with interlobular edema,(2) cutaneous pallor or icterus, (3) mottled atrophic livers, (4)gastric ulcers and (5) nephritis. At necropsy (Day 50), icterus,hepatitis, and nephritis were not noted in any groups. A gastric ulcerwas noted in one Group 7 pig, but lymphadenopathy was not specificallyexamined for. Based on the presence of lesions that were consistent withPCV2 infection, three groups had at least one pig tentatively diagnosedwith PCV2 (PMWS). Group 8, which received two doses of killed vaccine,had 20% tentatively diagnosed with PCV2, while Group 7 and Group 4 had13.3% and 6.7%, respectively, tentatively diagnosed with PCV2. The mean% lung lesion scores varied between groups at necropsy. Groups 1, 2, 3,4, 6 and 10 had low % lung lesion scores that ranged from 0.11±0.38% to0.90±0.15%. As expected, Group 9, the challenge control group, had thehighest mean % lung lesion score (10.81±23.27%). In four groups, themean % lung lesion scores were elevated due to one to three pigs in eachof these groups having very high lung lesion scores. The lung lesionswere red/purple and consolidated. Typically, lung lesions associatedwith PMWS are described as tan, non-collapsible with interlobular edema.The lung lesions noted in this study were either not associated withPCV2 infection or a second pulmonary infectious agent may have beenpresent. Within the context of this study, the % lung lesion scoresprobably do not reflect a true measure of the amount of lung infectiondue to PCV2. Likewise, tentative diagnosis of pneumonia may have beenover-utilized as well. Any pigs with lung lesions, some as small as0.10% were listed with a tentative diagnosis of pneumonia. In thisexample, there was no sufficient difference between groups with respectto gross lesions and % lung lesions on which to base vaccine efficacy.

IHC results showed the largest differences between groups. Group 1 (16μg rORF2—IMS 1314) had the lowest positive IHC results for PCV2 antigen(0%); while Groups 9 and 10 had the highest positive IHC results withincidence rates of 100% and 93.3% respectively. Groups 3, 5, 6 and 7,which received 16, 4, 1 or 0.25 μg of rORF2 antigen, respectively,adjuvanted with Carbopol, had IHC positive rates of 20%, 20%, 40% and46.7%, respectively. Group 2, which received two doses of 16 μg vORF2adjuvanted with Carbopol had an IHC positive rate of 6.7%, while Group 4which received only one dose of the same vaccine, had an IHC positiverate of 13.3%. Due to the objective nature of this test and the factthat IHC results correlated with expected results, IHC testing isprobably one of the best parameters on which to base vaccine efficacy.

Thus in one aspect of the present invention, the Minimum ProtectiveDosage (MPD) of PCV2 rORF2 antigen adjuvanted with Carbopol in the CDCDpig model in the face of a PCV2 challenge is determined Groups 3, 5, 6and 7 each received two doses of rORF2 antigen adjuvanted with Carbopol,but the level of rORF2 antigen varied for each group. Groups 3, 5, 6 and7 each received 16, 4, 1 or 0.25 μg of rORF2 antigen respectively. Ingeneral, decreasing the level of rORF2 antigen decreased PCV2 antibodytiters, and increased the mortality rate, mean % lung lesions and theincidence of IHC positive tissues. Of the four groups receiving varyinglevels of rORF2—Carbopol, Groups 3 and 5, which received two doses of 16or 4 μg of rORF2 antigen, respectively, each had an IHC positive rate ofonly 20%, and each had similar antibody titers. Overall, based on IHCpositive results, the minimum protective dosage of rORF2 antigenadministered twice is approximately 4 ng.

In another aspect of the present invention, the antigenicity ofrecombinant (rORF2) and VIDO R-1 (vORF2) PCV2 antigens were assessed.Group 2 received two doses of 16 ng vORF2 and Group 3 received two dosesof 16 μg rORF2. Both vaccines were adjuvanted with Carbopol. Bothvaccines were found to be safe and both had 0% mortality rate. Group 2had a PCV2 antibody titer of 2507 on Day 25, while Group 3 had a PCV2antibody titer of 1503. Group 3 had a lower mean % lung lesion scorethan Group 2 (0.11±0.38% vs. 0.90±0.15%), but Group 2 had a lower IHCpositive incidence rate that Group 3 (6.7% vs. 20%). Overall, bothvaccines had similar antigenicity, but vORF2 was associated withslightly better IHC results.

In yet another aspect of the present invention, the suitability of twodifferent adjuvants (Carbopol and IMS 1314) was determined Groups 1 and3 both received two doses of vaccine containing 16 ug of rORF2 antigen,but Group 1 received the antigen adjuvanted with IMS 1314 while Group 3received the antigen adjuvanted with Carbopol. Both groups hadessentially the same ADWG, essentially the same incidence of clinicalsigns post-challenge, the same mortality rate, and essentially the samemean % lung lesions; but Group 1 had an IHC positive rate of 0% whileGroup 3 had an IHC positive rate of 20%. However, Group 3, whichreceived the vaccine adjuvanted with Carbopol had higher IFAT PCV2titers on Days 25, 32 and 50 than Group 1, which received the vaccineadjuvanted with IMS 1314. Overall, although the PCV2 vaccine adjuvantedwith IMS 1314 did provide better IHC results, it did not provideoverwhelmingly better protection from PCV2 infection and did induceinjection site reaction. Whereas the PCV2 vaccine adjuvanted withCarbopol performed nearly as well as the IMS 1314 adjuvanted vaccine,but was not associated with any adverse events.

In still another aspect of the present invention, the feasibility ofPCV2 ORF2 as a lml, 1 dose product was determined Groups 2 and 4 bothreceived 16 μg of vORF2 vaccine adjuvanted with Carbopol on Day 0, butGroup 2 received a second dose on Day 14. Group 4 had a slightly higherADWG and a lower mean % lung lesions than Group 2, but Group 2 hadhigher IFAT PCV2 titers on Day 25, 32 and 50, and a slightly lowerincidence rate of IHC positive tissues. All other results for these twogroups were similar. Overall, one dose of vORF2 adjuvanted with Carbopolperformed similar to two doses of the same vaccine.

1-22. (canceled)
 23. A method for reducing or lessening the severity ofthe clinical symptoms associated with PCV2 infection and/or infectionscaused by Mycoplasma hyopneumoniae, Porcine Reproductive and RespiratorySyndrome Virus, or a combination thereof, comprising administering to apig in need thereof a single dose multivalent combination vaccinecomprising 4 μg to 400 μg recombinant PCV2 ORF2 protein per dose, atleast one further immunogenic active component against Mycoplasmahyopneumoniae, Porcine Reproductive and Respiratory Syndrome Virus, or acombination thereof, and at least one additional component selected fromthe group consisting of an effective amount of antimicrobial activeagent or an effective amount of stabilizing agent that increases theshelf-life of the vaccine, wherein reducing or lessening the severity ofthe clinical symptoms after the administration of said vaccine iscompared to those swine not receiving said vaccine and wherein saidclinical symptoms are selected from the group consisting of wasting,paleness of the skin, unthriftiness, respiratory distress, diarrhea,icterus, jaundice, lung lesions, nasal shedding, cough, diarrhea andcombinations thereof.
 24. The method of claim 23, wherein said PCV2 ORF2protein is a) a polypeptide selected from the group consisting of SEQ IDNO: 5, SEQ ID NO: 6, and SEQ ID NO: 11; or b) any polypeptide that is atleast 90% identical to the polypeptide of a).
 25. The method of claim23, wherein said vaccine comprising recombinant PCV2 ORF2 proteinfurther comprises an additional component selected from the groupconsisting of an inactivated viral vector, cell culture supernatant,BEI, sodium thiosulfate, carriers, adjuvants, media, viral inactivators,diluents, isotonic agents, immunomodulatory agents, antibiotics,pharmaceutical acceptable salts, and combinations thereof.
 26. Themethod of claim 23, wherein said single dose has a volume of at least 1ml.
 27. The method of claim 23, wherein said single dose has a volume of2 ml.
 28. The method of claim 23, wherein said vaccine is retainedwithin a container.
 29. The method of claim 23, wherein said furtherimmunogenic active component is Mycoplasma hyopneumoniae.
 30. The methodof claim 23, wherein said further immunogenic active component isPorcine Reproductive and Respiratory Syndrome Virus.
 31. The method ofclaim 23, wherein said further immunogenic active components areMycoplasma hyopneumoniae and Porcine Reproductive and RespiratorySyndrome Virus.
 32. The method of claim 31, wherein said single dosecombination vaccine comprises 3-10 logs of Porcine Reproductive andRespiratory Syndrome Virus.
 33. The method of claim 31, wherein saidsingle dose combination vaccine comprises the antigen included inIngelvac PRRS MLV.
 34. The method of claim 23, wherein said immunogenicactive component is an attenuated microorganism, including modified livevirus (MLV), a killed-microorganism or at least an immunological activepart of a microorganism.
 35. The method of claim 23, wherein saidimmunological active part of a microorganism is a protein-, sugar-, andor glycoprotein containing fraction of a microorganism that comprises atleast one antigen that induces or stimulates the immune response in ananimal to which said component is administered.
 36. The method of claim23, wherein the amount of PCV2 ORF2 protein in the vaccine is 4 μg to400 μg per dose.
 37. The method of claim 23, wherein the amount of PCV2ORF2 protein in the vaccine is 4 μg to 200 μg per dose.
 38. The methodof claim 23, wherein a single dose combination vaccine comprises theantigen included in Inglevac® CircoFLEX™.
 39. A method for inducing animmunological response in a pig comprising administering to the pig amultivalent combination vaccine comprising recombinant PCV2 ORF2protein, and at least one immunogenic active component againstMycoplasma hyopneumoniae, Porcine Reproductive and Respiratory SyndromeVirus, or a combination thereof, wherein said composition reduces theseverity of one or more clinical symptoms associated with PCV2 infectionand/or infections caused by Mycoplasma hyopneumoniae, and/or PorcineReproductive and Respiratory Syndrome Virus, or a combination thereof,after the administration of vaccine, wherein said one or more clinicalsymptoms are selected from the group consisting of wasting, paleness ofthe skin, unthriftiness, respiratory distress, diarrhea, icterus,jaundice, lung lesions, nasal shedding, cough, diarrhea and combinationsthereof.
 40. The method of claim 39, wherein said PCV2 ORF2 protein isa) a polypeptide selected from the group consisting of SEQ ID NO: 5, SEQID NO: 6, and SEQ ID NO: 11; or b) any polypeptide that is at least 90%identical to the polypeptide of a).
 41. The method of claim 39, whereinsaid vaccine comprising recombinant PCV2 ORF2 protein further comprisesan additional component selected from the group consisting of aninactivated viral vector, cell culture supernate, BEI, sodiumthiosulfate, carriers, adjuvants, media, viral inactivators, diluents,isotonic agents, immunomodulatory agents, antibiotics, pharmaceuticalacceptable salts, and combinations thereof.
 42. The method of claim 39,wherein said single dose has a volume of at least 1 ml.
 43. The methodof claim 39, wherein said single dose has a volume of 2 ml.
 44. Themethod of claim 39, wherein said vaccine is retained within a container.45. The method of claim 39, wherein said further immunogenic activecomponent is Mycoplasma hyopneumoniae.
 46. The method of claim 39,wherein said further immunogenic active components are Mycoplasmahyopneumoniae and Porcine Reproductive and Respiratory Syndrome Virus.47. The method of claim 46, wherein said single dose combination vaccinecomprises 3-10 logs of Porcine Reproductive and Respiratory SyndromeVirus.
 48. The method of claim 46, wherein said single dose combinationvaccine comprises the antigen included in Ingelvac PRRS MLV.
 49. Themethod of claim 39, wherein said immunogenic active component is anattenuated microorganism, including modified live virus (MLV), akilled-microorganism or at least an immunological active part of amicroorganism.
 50. The method of claim 39, wherein said immunologicalactive part of a microorganism is a protein-, sugar-, and orglycoprotein containing fraction of a microorganism that comprises atleast one antigen that induces or stimulates the immune response in ananimal to which said component is administered.
 51. The method of claim39, wherein the amount of PCV2 ORF2 protein in the vaccine is 4 μg to400 μg per dose.
 52. The method of claim 39, wherein the amount of PCV2ORF2 protein in the vaccine is 4 μg to 200 μg per dose.
 53. The methodof claim 39, wherein a single dose combination vaccine comprises theantigen included in Inglevac® CircoFLEX™.